Processes for preparation of 9,11- epoxy steroids and intermediates useful therein

ABSTRACT

Multiple novel reaction schemes, novel process steps and novel intermediates are provided for the synthesis of epoxymexrenone and other compounds of Formula I  
                 
 
     wherein:  
     —A—A— represents the group —CHR 4 —CHR 5 — or —CR 4 ═CR 5 — 
     R 3 , R 4  and R 5  are independently selected from the group consisting of hydrogen, halo, hydroxy, lower alkyl, lower alkoxy, hydroxyalkyl, alkoxyalkyl, hydroxycarbonyl, cyano, aryloxy,  
     R 1  represents an alpha-oriented lower alkoxycarbonyl or hydroxyalkyl radical,  
     —B—B— represents the group —CHR 6 —CHR 7 — or an alpha- or beta-oriented group:  
                 
 
      where R 6  and R 7  are independently selected from the group consisting of hydrogen, halo, lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl, hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl, cyano, aryloxy, and  
     R 8  and R 9  are independently selected from the group consisting of hydrogen, halo, lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl, hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl, cyano, aryloxy, or R 8  and R 9  together comprise a carbocyclic or heterocyclic ring structure, or R 8  or R 9  together with R 6  or R 7  comprise a carbocyclic or heterocyclic ring structure fused to the pentacyclic D ring.

BACKGROUND OF THE INVENTION

[0001] This invention relates to the novel processes for the preparationof 9,11-epoxy steroid compounds, especially those of the 20-spiroxaneseries and their analogs, novel intermediates useful in the preparationof steroid compounds, and processes for the preparation of such novelintermediates. Most particularly, the invention is directed to novel andadvantageous methods for the preparation of methyl hydrogen9,11α-epoxy-17α-hydroxy-3-oxopregn-4-ene-7α,21-dicarboxylate, γ-lactone(eplerenone; epoxymexrenone).

[0002] Methods for the preparation of 20-spiroxane series compounds aredescribed in U.S. Pat. No. 4,559,332. The compounds produced inaccordance with the process of the '332 patent have an open oxygencontaining ring E of the general formula:

[0003] in which

[0004] —A—A— represents the group —CH₂—CH₂— or —CH═CH—,

[0005] R¹ represents an α-oriented lower alkoxycarbonyl orhydroxycarbonyl radical.

[0006] —B—B— represents the group —CH₂—CH₂— or an α- or β-oriented group

[0007] R⁶ and R⁷ being hydrogen

[0008] X represents two hydrogen atoms or oxo,

[0009] Y¹ and Y² together represent the oxygen bridge —O—, or

[0010] Y¹ represents hydroxy, and

[0011] Y² represents hydroxy, lower alkoxy or, if X represents H₂, alsolower alkanoyloxy,

[0012] and salts of such compounds in which X represents oxo and Y²represents hydroxy, that is to say of corresponding17β-hydroxy-21-carboxylic acids.

[0013] U.S. Pat. No. 4,559,332 describes a number of methods for thepreparation of epoxymexrenone and related compounds of Formula IA. Theadvent of new and expanded clinical uses for epoxymexrenone create aneed for improved processes for the manufacture of this and otherrelated steroids.

SUMMARY OF THE INVENTION

[0014] The primary object of the present invention is the provision ofimproved processes for the preparation of epoxymexrenone, other20-spiroxanes and other steroids having common structural features.Among the particular objects of the invention are: to provide animproved process that produces products of Formula IA and other relatedcompounds in high yield; the provision of such a process which involvesa minimum of isolation steps; and the provision of such a process whichmay be implemented with reasonable capital expense and operated atreasonable conversion cost.

[0015] Accordingly, the present invention is directed to a series ofsynthesis schemes for epoxymexrenone; intermediates useful in themanufacture of eplerenone; and syntheses for such novel intermediates.

[0016] The novel synthesis schemes are described in detail in theDescription of Preferred Embodiments. Among the novel intermediates ofthis invention are those described immediately below.

[0017] A compound of Formula IV corresponds to the structure:

[0018] wherein:

[0019] —A—A— represents the group —CHR⁴—CHR⁵— or —CR⁴═CR⁵—

[0020] R³, R⁴ and R⁵ are independently selected from the groupconsisting of hydrogen, halo, hydroxy, lower alkyl, lower alkoxy,hydroxyalkyl, alkoxyalkyl, hydroxy carbonyl, cyano, aryloxy,

[0021] R¹ represents an alpha-oriented lower alkoxycarbonyl orhydroxycarbonyl radical,

[0022] R² is an 11α-leaving group the abstraction of which is effectivefor generating a double bond between the 9- and 11-carbon atoms;

[0023] —B—B— represents the group —CHR⁶—CHR⁷— or an alpha- orbeta-oriented group:

[0024]  where R⁶ and R⁷ are independently selected from the groupconsisting of hydrogen, halo, lower alkoxy, acyl, hydroxalkyl,alkoxyalkyl, hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl,cyano, aryloxy, and

[0025] R⁸ and R⁹ are independently selected from the group consisting ofhydrogen, halo, lower alkoxy, acyl, hydroxalkyl, alkoxyalkyl,hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl, cyano, aryloxy, orR⁸ and R⁹ together comprise a carbocyclic or heterocyclic ringstructure, or R⁸ or R⁹ together with R⁶ or R⁷ comprise a carbocyclic orheterocyclic ring structure fused to the pentacyclic D ring.

[0026] A compound of Formula IVA corresponds to Formula IV wherein R⁸and R⁹ together with the ring carbon to which they are attached form thestructure:

[0027] where X, Y¹, Y² and C(17) are as defined above.

[0028] A compound of Formula IVB corresponds to Formula IVA wherein R⁸and R⁹ together form the structure of Formula XXXIII:

[0029] Compounds of Formulae IVC, IVD and IVE, respectively, correspondto any of Formula IV, IVA, or IVB wherein each of —A—A— and —B—B— is—CH₂—CH₂—, R³ is hydrogen, and R¹ is alkoxycarbonyl, preferablymethoxycarbonyl. Compounds within the scope of Formula IV may beprepared by reacting a lower alkylsulfonylating or acylating reagent, ora halide generating agent, with a corresponding compound within thescope of Formula V.

[0030] A compound of Formula V corresponds to the structure:

[0031] wherein —A—A—, —B—B—, R¹, R³, R⁸ and R⁹ are as defined in FormulaIV.

[0032] A compound of Formula VA corresponds to Formula V wherein R⁸ andR⁹ with the ring carbon to which they are attached together form thestructure:

[0033] where X, Y¹, Y² and C(17) are as defined above.

[0034] A compound of Formula VB corresponds to Formula VA wherein R⁸ andR⁹ together form the structure of Formula XXXIII:

[0035] Compounds of Formulae VC, VD and VE, respectively, correspond toany of Formula V, VA, or VB wherein each of —A—A— and —B—B— is—CH₂—CH₂—, R³ is hydrogen, and R¹ is alkoxycarbonyl, preferablymethoxycarbonyl. Compounds within the scope of Formula V may be preparedby reacting an alkali metal alkoxide with a corresponding compound ofFormula VI.

[0036] A compound of Formula VI corresponds to the structure:

[0037] wherein —A—A—, —B—B—, R³, R⁸ and R⁹ are as defined in Formula IV.

[0038] A compound of Formula VIA corresponds to Formula VI wherein R⁸and R⁹ together with the ring carbon to which they are attached form thestructure:

[0039] where X, Y¹, Y² and C(17) are as defined above.

[0040] A compound of Formula VIB corresponds to Formula VIA wherein R⁸and R⁹ together form the structure of Formula XXXIII:

[0041] Compounds of Formulae VIC, VID and VIE, respectively, correspondto any of Formula VI, VIA, or VIB wherein each of —A—A— and —B—B— is—CH₂—CH₂—, and R³ is hydrogen. Compounds of Formula VI, VIA, VIB and VICare prepared by hydrolyzing a compound corresponding to Formula VII,VIIA, VIIB or VIIC, respectively.

[0042] A compound of Formula VII corresponds to the structure:

[0043] wherein —A—A—, —B—B—, R³, R⁸ and R⁹ are as defined in Formula IV.

[0044] A compound of Formula VIIA corresponds to Formula VII wherein R⁸and R⁹ together with the ring carbon to which they are attached form thestructure:

[0045] where X, Y¹, Y² and C(17) are as defined above.

[0046] A compound of Formula VIIB corresponds to Formula VIIA wherein R⁸and R⁹ together form the structure of Formula XXXIII:

[0047] Compounds of Formulae VIIC, VIID and VIIE, respectively,correspond to any of Formula VII, VIIA, or VIIB wherein each of —A—A—and —B—B— is —CH₂—CH₂—, and R³ is hydrogen. A compound within the scopeof Formula VII may be prepared by cyanidation of a compound within thescope of Formula VIII.

[0048] A compound of Formula VIII corresponds to the structure:

[0049] wherein —A—A—, —B—B—, R³, R⁸ and R⁹ are as defined in Formula IV.

[0050] A compound of Formula VIIIA corresponds to Formula VIII whereinR⁸ and R⁹ together with the ring carbon to which they are attached formthe structure:

[0051] where X, Y¹, Y² and C(17) are as defined above.

[0052] A compound of Formula VIIIB corresponds to Formula VIIIA whereinR⁸ and R⁹ together form the structure of Formula XXXIII:

[0053] Compounds of Formulae VIIIC, VIIID and VIIIE, respectively,correspond to any of Formula VIII, VIIIA, or VIIIB wherein each of —A—A—and —B—B— is —CH₂—CH₂—, and R³ is hydrogen. Compounds within the scopeof Formula VIII are prepared by oxidizing a substrate comprising acompound of Formula XXX as described hereinbelow by fermentationeffective for introducing an 11-hydroxy group into the substrate inα-orientation.

[0054] A compound of Formula XIV corresponds to the structure:

[0055] wherein —A—A—, —B—B—, R³, R⁸ and R⁹ are as defined in Formula IV.

[0056] A compound of Formula XIVA corresponds to Formula XIV wherein R⁸and R⁹ together with the ring carbon to which they are attached form thestructure:

[0057] where X, Y¹, Y² and C(17) are as defined above.

[0058] A compound of Formula XIV corresponds to Formula XIVA wherein R⁸and R⁹ together with the ring carbon to which they are attached form thestructure of Formula XXXIII:

[0059] Compounds of Formulae XIVC, XIVD and XIVE, respectively,correspond to any of Formula XIV, XIVA, or XIVB wherein each of —A—A—and —B—B— is —CH₂—CH₂—, and R³ is hydrogen. Compounds within the scopeof Formula XIV can be prepared by hydrolysis of a corresponding compoundwithin the scope of Formula XV.

[0060] A compound of Formula XV corresponds to the structure:

[0061] wherein —A—A—, —B—B—, R³, R⁸ and R⁹ are as defined in Formula IV.

[0062] A compound of Formula XVA corresponds to Formula XV wherein R⁸and R⁹ together with the ring carbon to which they are attached form thestructure:

[0063] where X, Y¹, Y² and C(17) are as defined above.

[0064] A compound of Formula XVB corresponds to Formula XVA wherein R⁸and R⁹ together with the ring carbon to which they are attached form thestructure of Formula XXXIII:

[0065] Compounds of Formulae XVC, XVD and XVE, respectively, correspondto any of Formula XV, XVA, or XVB wherein each of —A—A— and —B—B— is—CH₂—CH₂—, and R³ is hydrogen. Compounds within the scope of Formula XVcan be prepared by cyanidation of a corresponding compound within thescope of Formula XVI.

[0066] A compound of Formula XXI corresponds to the structure:

[0067] wherein —A—A—, —B—B—, R³, R⁸ and R⁹ are as defined in Formula IV.

[0068] A compound of Formula XXIA corresponds to Formula XXI wherein R⁸and R⁹ together with the ring carbon to which they are attached form thestructure:

[0069] where X, Y¹, Y² and C(17) are as defined above.

[0070] A compound of Formula XXIB corresponds to is Formula XXIA whereinR⁸ and R⁹ together form the structure of Formula XXXIII:

[0071] Compounds of Formulae XXIC, XXID and XXIE, respectively,correspond to any of Formula XXI, XXIA, or XXIB wherein each of —A—A—and —B—B— is —CH₂—CH₂—, and R³ is hydrogen. Compounds within the scopeof Formula XXI may be prepared by hydrolyzing a corresponding compoundwithin the scope of Formula XXII.

[0072] A compound of Formula XXII corresponds to the structure:

[0073] wherein —A—A—, —B—B—, R³, R⁸ and R⁹ are as defined in Formula IV.

[0074] A compound of Formula XXIIA corresponds to Formula XXII whereinR⁸ and R⁹ together with the ring carbon to which they are attached formthe structure:

[0075] where X, Y¹, Y² and C(17) are as defined above.

[0076] A compound of Formula XXIIB corresponds to Formula XXIIA whereinR⁸ and R⁹ together form the structure of Formula XXXIII:

[0077] Compounds of Formulae XXIIC, XXIID and XXIIE, respectively,correspond to any of Formula XXII, XXIIA, or XXIIB wherein each of —A—A—and —B—B— is —CH₂—CH₂—, and R³ is hydrogen. Compounds within the scopeof Formula XXII may be prepared by cyanidation of a compound within thescope of Formula XXIII.

[0078] A compound of Formula XXIII corresponds to the structure:

[0079] wherein —A—A—, —B—B—, R³, R⁸ and R⁹ are as defined in Formula IV.

[0080] A compound of Formula XXIIIA corresponds to Formula XXIII whereinR⁸ and R⁹ together with the ring carbon to which they are attached formthe structure:

[0081] where X, Y¹, Y² and C(17) are as defined above.

[0082] A compound of Formula XXIIIB corresponds to Formula XXIIIAwherein R⁸ and R⁹ together form the structure of Formula XXXIII:

[0083] Compounds of Formulae XXIIIC, XXIIID and XXIIIE, respectively,correspond to any of Formula XXIII, XXIIIA, or XXIIIB wherein each of—A—A— and —B—B— is —CH₂—CH₂—, and R³ is hydrogen. Compounds within thescope of Formula XXIII can be prepared by oxidation of a compound ofFormula XXIV, as described hereinbelow.

[0084] A compound of Formula 104 corresponds to the structure:

[0085] wherein —A—A—, —B—B—, and R³ are as defined in Formula IV, andR¹¹ is C₁ to C₄ alkyl.

[0086] A compound of Formula 104A corresponds to Formula 104 whereineach of —A—A— and —B—B— is —CH₂—CH₂—, and R³ is hydrogen. Compoundswithin the scope of Formula 104 may be prepared by thermal decompositionof a compound of Formula 103.

[0087] A compound of Formula 103 corresponds to the structure:

[0088] wherein —A—A—, —B—B—, R³ and R¹¹ are as defined in Formula 104.

[0089] A compound of Formula 103A corresponds to Formula 103 whereineach of —A—A— and —B—B— is —CH₂—CH₂—, and R³ is hydrogen. Compoundswithin the scope of Formula 103 may be prepared by reaction of acorresponding compound of Formula 102 with a dialkyl malonate in thepresence of a base such as an alkali metal alkoxide.

[0090] A compound of Formula 102 corresponds to the structure:

[0091] wherein —A—A—, —B—B—, R³ and R¹¹ are as defined in Formula 104.

[0092] A compound of Formula 102A corresponds to Formula 102 whereineach of —A—A— and —B—B— is —CH₂—CH₂—, and R³ is hydrogen. Compoundswithin the scope of Formula 102 may be prepared by reaction of acorresponding compound of Formula 101 with a trialkyl sulfonium compoundin the presence of a base.

[0093] A compound of Formula 101 corresponds to the structure:

[0094] wherein —A—A—, —B—B—, R³ and R¹¹ are as defined in Formula 104.

[0095] A compound of Formula 101A corresponds to Formula 101 whereineach of —A—A— and —B—B— is —CH₂—CH₂—, and R³ is hydrogen. Compoundswithin the scope of Formula 101 may be prepared by reaction of11α-hydroxyandrostene-3,17-dione or other compound of Formula XXXVI witha trialkyl orthoformate in the presence of an acid.

[0096] Based on the disclosure of specific reaction schemes as set outhereinbelow, it will be apparent which of these compounds have thegreatest utility relative to a particular reaction scheme. Use of thecompounds of this invention are useful as intermediates forepoxymexrenone and other steroids.

[0097] Other objects and features will be in part apparent and in partpointed out hereinafter.

BRIEF DESCRIPTION OF THE DRAWINGS

[0098]FIG. 1 is a schematic flow sheet of a process for thebioconversion of canrenone or a canrenone derivative to thecorresponding 11α-hydroxy compound;

[0099]FIG. 2 is a schematic flow sheet of a preferred process for thebioconversion of 11-α-hydroxylation of canrenone and canrenonederivatives;

[0100]FIG. 3 is a schematic flow sheet of a particularly preferredprocess for the bioconversion of 11-α-hydroxylation of canrenone andcanrenone derivatives;

[0101]FIG. 4 shows the particle size distribution for canrenone asprepared in accordance with the process of FIG. 2; and

[0102]FIG. 5 shows the particle size distribution for canrenone assterilized in the transformation fermenter in accordance with theprocess of FIG. 3.

[0103] Corresponding reference characters indicate corresponding partsthroughout the drawings.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0104] In accordance with the present invention, various novel processschemes have been devised for the preparation of epoxymexrenone andother compounds corresponding Formula I:

[0105] wherein:

[0106] —A—A— represents the group —CHR⁴—CHR⁵— or —CR⁴═CR⁵—

[0107] R³, R⁴ and R⁵ are independently selected from the groupconsisting of hydrogen, halo, hydroxy, lower alkyl, lower alkoxy,hydroxyalkyl, alkoxyalkyl, hydroxycarbonyl, cyano, aryloxy,

[0108] R¹ represents an alpha-oriented lower alkoxycarbonyl orhydroxyalkyl radical,

[0109] —B—B— represents the group —CHR⁶—CHR⁷— or an alpha- orbeta-oriented group:

[0110]  where R⁶ and R⁷ are independently selected from the groupconsisting of hydrogen, halo, lower alkoxy, acyl, hydroxyalkyl,alkoxyalkyl, hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl,cyano, aryloxy, and

[0111] R⁸ and R⁹ are independently selected from the group consisting ofhydrogen, halo, lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl,hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl, cyano, aryloxy, orR⁸ and R⁹ together comprise a carbocyclic or heterocyclic ringstructure, or R⁸ or R⁹ together with R⁶ or

[0112] R⁷ comprise a carbocyclic or heterocyclic ring structure fused tothe pentacyclic D ring.

[0113] Unless stated otherwise, organic radicals referred to as “lower”in the present disclosure contain at most 7, and preferably from 1 to 4,carbon atoms.

[0114] A lower alkoxycarbonyl radical is preferably one derived from analkyl radical having from 1 to 4 carbon atoms, such as methyl, ethyl,propyl, isopropyl, butyl, isobutyl, sec.-butyl and tert.-butyl;especially preferred are methoxycarbonyl, ethoxycarbonyl andisopropoxycarbonyl. A lower alkoxy radical is preferably one derivedfrom one of the above-mentioned C₁-C₄ alkyl radicals, especially from aprimary C₁-C₄ alkyl radical; especially preferred is methoxy. A loweralkanoyl radical is preferably one derived from a straight-chain alkylhaving from 1 to 7 carbon atoms; especially preferred are formyl andacetyl.

[0115] A methylene bridge in the 15,16-position is preferablyβ-oriented.

[0116] A preferred class of compounds that may be produced in accordancewith the methods of the invention are the 20-spiroxane compoundsdescribed in U.S. Pat. No. 4,559,332, i.e., those corresponding toFormula IA:

[0117] where:

[0118] —A—A— represents the group —CH₂—CH₂— or —CH═CH—,

[0119] —B—B— represents the group —CH₂—CH₂— or an alpha- orbeta-oriented group of Formula IIIA:

[0120] R¹ represents an alpha-oriented lower alkoxycarbonyl orhydroxycarbonyl radical, X represents two hydrogen atoms, oxo or ═S

[0121] Y¹ and Y² together represent the oxygen bridge —O—, or

[0122] Y¹ represents hydroxy, and

[0123] Y² represents hydroxy, lower alkoxy or, if X represents H₂, alsolower alkanoyloxy,

[0124] Preferably, 20-spiroxane compounds produced by the novel methodsof the invention are those of Formula I in which Y¹ and Y² togetherrepresent the oxygen bridge —O—.

[0125] Especially preferred compounds of the formula I are those inwhich X represents oxo.

[0126] Of compounds of the 20-spiroxane compounds of Formula IA in whichX represents oxo there are most especially preferred those in which Y¹together with Y² represents the oxygen bridge —O—.

[0127] As already mentioned, 17β-hydroxy-21-carboxylic acid may also bein the form of their salts. There come into consideration especiallymetal and ammonium salts, such as alkali metal and alkaline earth metalsalts, for example sodium, calcium, magnesium and, preferably,potassium, salts, and ammonium salts derived from ammonia or a suitable,preferably physiologically tolerable, organic nitrogen-containing base.As bases there come into consideration not only amines, for examplelower alkylamines (such as triethylamine), hydroxy-lower alkylamines[such as 2-hydroxyethylamine, di-(2-hydroxyethyl)-amine ortri-(2-hydroxyethyl)-amine], cycloalkylamines (such asdicyclohexylamine) or benzylamines (such as benzylamine andN,N′-dibenzylethylenediamine), but also nitrogen-containing heterocycliccompounds, for example those of aromatic character (such as pyridine orquinoline) or those having an at least partially saturated heterocyclicring (such as N-ethylpiperidine, morpholine, piperazine orN,N′-dimethylpiperazine).

[0128] Also included amongst preferred compounds are alkali metal salts,especially potassium salts, of compounds of the formula IA in which R¹represents alkoxycarbonyl, with X representing oxo and each of Y¹ and Y²representing hydroxy.

[0129] Especially preferred compounds of the formula I and IA are, forexample, the following:

[0130] 9α,11α-epoxy-7α-methoxycarbonyl-20-spirox-4-ene-3,21-dione,

[0131] 9α,11α-epoxy-7α-ethoxycarbonyl-20-spirox-4-ene-3,21-dione,

[0132] 9α,11α-epoxy-7α-isopropoxycarbonyl-20-spirox-4-ene-3,21-dione,

[0133] and the 1,2-dehydro analogue of each of the compounds,

[0134] 9α,11α-epoxy-6α,7α-methylene-20-spirox-4-ene-3,21-dione,

[0135] 9α,11α-epoxy-6β,7β-methylene-20-spirox-4-ene-3,21-dione,

[0136]9α,11α-epoxy-6β,7β;15β,16β-bismethylene-20-spirox-4-ene-3,21-dione,

[0137] and the 1,2-dehydro analogue of each of these compounds,

[0138]9α,11α-epoxy-7α-methoxycarbonyl-17β-hydroxy-3-oxo-pregn-4-ene-21-carboxylicacid,

[0139]9α,11α-epoxy-7α-ethoxycarbonyl-17β-hydroxy-3-oxo-pregn-4-ene-21-carboxylicacid,

[0140]9α,11α-epoxy-7α-isopropoxycarbonyl-17β-hydroxy-3-oxo-pregn-4-ene-21-carboxylicacid,

[0141] 9α,11α-epoxy-17β-hydroxy-6α,7α-methylene-3-oxo-pregn-4-ene-21-carboxylic acid,

[0142]9α,11α-epoxy-17β-hydroxy-6β,7β-methylene-3-oxo-pregn-4-ene-21-carboxylicacid,

[0143]9α,11α-epoxy-17β-hydroxy-6β,7β;15β,16β-bismethylene-3-oxo-pregn-4-ene-21-carboxylicacid, and alkali metal salts, especially the potassium salt or ammoniumof each of these acids, and also a corresponding 1,2-dehydro analogue ofeach of the mentioned carboxylic acids or of a salt thereof.

[0144]9α,11α-epoxy-15β,16β-methylene-3,21-dioxo-20-spirox-4-ene-7α-carboxylicacid methyl ester, ethyl ester and isopropyl ester,

[0145]9α,11α-epoxy-1565β,16β-methylene-3,21-dioxo-20-spiroxa-1,4-diene-7α-carboxylicacid methyl ester,

[0146] ethyl ester and isopropyl ester,

[0147] and also 9α,11α-epoxy-3-oxo-20-spirox-4-ene-7α-carboxylic acidmethyl ester, ethyl ester and isopropyl ester,

[0148] 9α,11α-epoxy-6β,6β-methylene-20-spirox-4-en-3-one,

[0149] 9α,11α-epoxy-6β,7β;15β,16β-bismethylene-20-spirox-4-en-3-one,

[0150] and also9α,11α-epoxy,17β-hydroxy-17α(3-hydroxy-propyl)-3-oxo-androst-4-ene-7α-carboxylicacid methyl ester, ethyl ester and isopropyl ester,

[0151] 9α,11α-epoxy,17β-hydroxy-17α-(3-hydroxypropyl)-6α,7α-methylene-androst-4-en-3-one,

[0152] 9α,11α-epoxy-17β-hydroxy-17α-(3-hydroxypropyl)-6β,7β-methylene-androst-4-en-3-one,

[0153] 9α,11α-epoxy-17β-hydroxy-17α-(3-hydroxypropyl)-6β,7β;15β,16β-bismethylene-androst-4-en-3-one,

[0154] including 17α-(3-acetoxypropyl) and 17α-(3-fromyloxypropyl)analogues of the mentioned androstane compounds,

[0155] and also 1,2-dehydro analogues of all the mentioned compounds ofthe androst-4-en-3-one and 20-spirox-4-en-3-one series.

[0156] The chemical names of the compounds of the Formulae I and IA, andof analogue compounds having the same characteristic structuralfeatures, are derived according to current nomenclature in the followingmanner: for compounds in which Y¹ together with Y² represents —O—, from20-spiroxane (for example a compound of the formula IA in which Xrepresents oxo and Y¹ together with Y² represents —O— is derived from20-spiroxan-21-one); for those in which each of Y¹ and Y² representshydroxy and X represents oxo, from17β-hydroxy-17α-pregnene-21-carboxylic acid; and for those in which eachof Y¹ and Y² represents hydroxy and X represents two hydrogen atoms,from 17β-hydroxy-17α-(3-hydroxypropyl)-androstane. Since the cyclic andopen-chain forms, that is to say lactones and 17β-hydroxy-21-carboxylicacids and their salts, respectively, are so closely related to eachother that the latter may be considered merely as a hydrated form of theformer, there is to be understood hereinbefore and hereinafter, unlessspecifically stated otherwise, both in end products of the formula I andin starting materials and intermediates of analogous structure, in eachcase all the mentioned forms together.

[0157] In accordance with the invention, several separate processschemes have been devised for the preparation of compounds of Formula Iin high yield and at reasonable cost. Each of the synthesis schemesproceeds through the preparation of a series of intermediates. A numberof these intermediates are novel compounds, and the methods ofpreparation of these intermediates are novel processes.

[0158] Scheme 1 (Starting With Canrenone or Related Material)

[0159] One preferred process scheme for the preparation of compounds ofFormula I advantageously begins with canrenone or a related startingmaterial corresponding to Formula XIII

[0160] wherein

[0161] —A—A— represents the group —CHR⁴—CHR⁵— or —CR⁴═CR⁵—

[0162] R³, R⁴ and R⁵ are independently selected from the groupconsisting of hydrogen, halo, hydroxy, lower alkyl, lower alkoxy,hydroxyalkyl, alkoxyalkyl, hydroxycarbonyl, cyano, aryloxy,

[0163] —B—B— represents the group —CHR⁶—CHR⁷— or an alpha- orbeta-oriented group:

[0164]  where R⁶ and R⁷ are independently selected from the groupconsisting of hydrogen, halo, lower alkoxy, acyl, hydroxyalkyl,alkoxyalkyl, hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl,cyano, aryloxy, and

[0165] R⁸ and R⁹ are independently selected from the group consisting ofhydrogen, halo, lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl,hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl, cyano, aryloxy orR⁸ and R⁹ together comprise a carbocyclic or heterocyclic ringstructure, or R⁸ and R⁹ together with R⁶ or R⁷ comprise a carbocyclic orheterocyclic ring structure fused to the pentacyclic D ring. Using abioconversion process of the type illustrated in FIGS. 1 and 2, an11-hydroxy group of α-orientation is introduced in the compound ofFormula XIII, thereby producing a compound of Formula VIII:

[0166] where —A—A—, —B—B—, R³, R⁸ and R⁹ are as defined above.Preferably, the compound of Formula XIII has the structure

[0167] and the 11α-hydroxy product has the structure

[0168] in each of which

[0169] —A—A— represents the group —CH₂—CH₂— or —CH═CH—,

[0170] —B—B— represents the group —CH₂—CH₂— or an alpha- orbeta-oriented group:

[0171] X represents two hydrogen atoms, oxo or ═S,

[0172] Y¹ and Y² together represent the oxygen bridge —O—, or

[0173] Y¹ represents hydroxy, and

[0174] Y² represents hydroxy, lower alkoxy or, if X represents H₂, alsolower alkanoyloxy, and salts of compounds in which X represents oxo andY² represents hydroxy-, and the compound of Formula VIII produced in thereaction corresponds to Formula VIIIA

[0175] wherein —A—A—, —B—B—, Y¹, Y², and X are as defined in FormulaXXXA. More preferably, R⁸ and R⁹ together form the 20-spiroxanestructure:

[0176] —A—A— and —B—B— are each —CH₂—CH₂—, and R³ is hydrogen.

[0177] Among the preferred organisms that can be used in thishydroxylation step are Aspergillus ochraceus NRRL 405, Aspergillusochraceus ATCC 18500, Aspergillus niger ATCC 16888 and ATCC 26693,Aspergillus nidulans ATCC 11267, Rhizopus oryzae ATCC 11145, Rhizopusstolonifer ATCC 6227b, Streptomyces fradiae ATCC 10745, Bacillusmegaterium ATCC 14945, Pseudomonas cruciviae ATCC 13262, andTrichothecium roseum ATCC 12543. Other preferred organisms includeFusarium oxysporum f.sp.cepae ATCC 11171 and Rhizopus arrhizus ATCC11145.

[0178] Other organisms that have exhibited activity for this reactioninclude Absidia coerula ATCC 6647, Absidia glauca ATCC 22752,Actinomucor elegans ATCC 6476, Aspergillus flavipes ATCC 1030,Aspergillus fumigatus ATCC 26934, Beauveria bassiana ATCC 7159 and ATCC13144, Botryosphaeria obtusa IMI 038560, Calonectria decora ATCC 14767,Chaetomium cochliodes ATCC 10195, Corynespora cassiicola ATCC 16718,Cunninghamella blakesleeana ATCC 8688a, Cunninghamella echinulata ATCC3655, Cunninghamella elegans ATCC 9245, Curvularia clavata ATCC 22921,Curvularia lunata ACTT 12071, Cylindrocarpon radicicola ATCC 1011,Epicoccum humicola ATCC 12722, Gongronella butleri ATCC 22822, Hypomyceschrysospermus, Mortierella isabellina ATCC 42613, Mucor mucedo ATCC4605, Mucor griseo-cyanus ATCC 1207A, Myrothecium verrucaria ATCC 9095,Nocardia corallina, Paecilomyces carneus ATCC 46579, Penicillum patulumATCC 24550, Pithomyces atro-olivaceus IFO 6651, Pithomyces cynodontisATCC 26150, Pycnosporium sp. ATCC 12231, Saccharopolyspora erythrae ATCC11635, Sepedonium chrysospermum ATCC 13378, Stachylidium bicolor ATCC12672, Streptomyces hygroscopicus ATCC 27438, Streptomyces purpurascensATCC 25489, Syncephalastrum racemosum ATCC 18192, Thamnostylum piriformeATCC 8992, Thielavia terricola ATCC 13807, and Verticillium theobromaeATCC 12474.

[0179] Additional organisms that may be expected to show activity forthe 11α-hydroxylation include Cephalosporium aphidicola (Phytochemistry(1996), 42(2), 411-415), Cochliobolus lunatas (J. Biotechnol. (1995),42(2), 145-150), Tieghemella orchidis (Khim.-Farm.Zh. (1986), 20(7),871-876), Tieghemella hyalospora (Khim.-Farm.Zh. (1986), 20(7),871-876), Monosporium olivaceum (Acta Microbiol. Pol., Ser. B. (1973),5(2), 103-110), Aspergillus ustus (Acta Microbiol. Pol., Ser. B. (1973),5(2), 103-110), Fusarium graminearum (Acta Microbiol. Pol., Ser. B.(1973), 5(2), 103-110), Verticillium glaucum (Acta Microbiol. Pol., Ser.B. (1973), 5(2), 103-110), and Rhizopus nigricans (J. Steroid Biochem.(1987), 28(2), 197-201).

[0180] Preparatory to production scale fermentation for hydroxylation ofcanrenone or other substrates of Formula XIII, an inoculum of cells isprepared in a seed fermentation system comprising a seed fermenter, or aseries of two or more seed fermenters. A working stock spore suspensionis introduced into the first seed fermenter, together with a nutrientsolution for growth of cells. If the volume of inoculum desired orneeded for production exceeds that produced in the first seed fermenter,the inoculum volume may be progressively and geometrically amplified byprogression through the remaining fermenters in the seed fermentationtrain. Preferably, the inoculum produced in the seed fermentation systemis of sufficient volume and viable cells for achieving rapid initiationof reaction in the production fermenter, relatively short productionbatch cycles, and high production fermenter activity. Whatever thenumber of vessels in a train of seed fermenters, the second andsubsequent seed fermenters are preferably sized so that the extent ofdilution at each step in the train is essentially the same. The initialdilution of inoculum in each seed fermenter can be approximately thesame as the dilution in the production fermenter. Canrenone or otherFormula XIII substrate is charged to the production fermenter along withinoculum and nutrient solution, and the hydroxylation reaction conductedthere.

[0181] The spore suspension charged to the seed fermentation system isfrom a vial of working stock spore suspension taken from a plurality ofvials constituting a working stock cell bank that is stored undercryogenic conditions prior to use. The working stock cell bank is inturn derived from a master stock cell bank that has been prepared in thefollowing manner. A spore specimen obtained from an appropriate source,e.g., ATCC, is initially suspended in an aqueous medium such as, forexample, saline solution, nutrient solution or a surfactant solution,(e.g., a nonionic surfactant such as Tween 20 at a concentration ofabout 0.001% by weight), and the suspension distributed among cultureplates, each plate bearing a solid nutrient mixture, typically based ona non-digestible polysaccharide such as agar, where the spores arepropagated. The solid nutrient mixture preferably contains between about0.5% and about 5% by weight glucose, between about 0.05% and about 5% byweight of a nitrogen source, e.g., peptone, between about 0.05% andabout 0.5% by weight of a phosphorus source, e.g., an ammonium or alkalimetal phosphate such as dipotassium hydrogen phosphate, between about0.25% and about 2.5% by weight yeast lysate or extract (or other aminoacid source such as meat extract or brain heart infusion), between about1% and about 2% by weight agar or other non-digestible polysaccharide.Optionally, the solid nutrient mixture may further comprise and/orcontain between about 0.1% and about 5% by weight malt extract. The pHof the solid nutrient mixture is preferably between about 5.0 and about7.0, adjusted as required by alkali metal hydroxide or orthophosphoricacid. Among useful solid growth media are the following:

[0182] 1. Solid Medium #1: 1% glucose, 0.25% yeast extract, 0.3% K₂HPO₄and 2% agar (Bacto); pH adjusted to 6.5 with 20% NaOH.

[0183] 2. Solid Medium #2: 2% peptone (Bacto), 1% yeast extract (Bacto),2% glucose and 2% agar (Bacto); pH adjusted to 5 with 10% H₃PO₄.

[0184] 3. Solid Medium #3: 0.1% peptone (Bacto), 2% malt extract(Bacto), 2% glucose and 2% agar (Bacto); pH as is 5.3.

[0185] 4. Liquid Medium: 5% blackstrap molasses, 0.5% cornsteep liquor,0.25% glucose, 0.25% NaCl and 0.5% KH₂PO₄, pH adjusted to 5.8.

[0186] 5. Difco Mycological agar (low pH).

[0187] The number of agar plates used in the development of a masterstock cell bank can be selected with a view to future demands for masterstock, but typically about 15 to about 30 plates are so prepared. Aftera suitable period of growth, e.g., 7 to 10 days, the plates are scrapedin the presence of an aqueous vehicle, typically saline or buffer, forharvesting the spores, and the resulting master stock suspension isdivided among small vials, e.g., one ml. in each of a plurality of 1.5ml vials. To prepare a working stock spore suspension for use inresearch or production fermentation operations, the contents of one ormore of these second generation master stock vials can be distributedamong and incubated on agar plates in the manner described above for thepreparation of master stock spore suspension. Where routinemanufacturing operations are contemplated, as many as 100 to 400 platesmay be used to generate second generation working stock. Each plate isscraped into a separate working stock vial, each vial typicallycontaining one ml of the inoculum produced. For permanent preservation,both the master stock suspension and the second generation productioninoculum are advantageously stored in the vapor space of a cryogenicstorage vessel containing liquid N₂ or other cryogenic liquid.

[0188] In the process illustrated in FIG. 1, aqueous growth medium isprepared which includes a nitrogen source such as peptone, a yeastderivative or equivalent, glucose, and a source of phosphorus such as aphosphate salt. Spores of the microorganism are cultured in this mediumin the seed fermentation system. The preferred microorganism isAspergillus ochraceus NRRL 405 (ATCC 18500). The seed stock so producedis then introduced into the production fermenter together with thesubstrate of Formula XIII. The fermentation broth is agitated andaerated for a time sufficient for the reaction to proceed to the desireddegree of completion.

[0189] The medium for the seed fermenter preferably comprises an aqueousmixture which contains: between about 0.5% and about 5% by weightglucose, between about 0.05% and about 5% by weight of a nitrogensource, e.g., peptone, between about 0.05% and about 0.5% by weight of aphosphorus source, e.g., an ammonium or alkali metal phosphate such asammonium phosphate monobasic or dipotassium hydrogen phosphate, betweenabout 0.25% and about 2.5% by weight yeast lysate or extract (or otheramino acid source such as distiller's solubles), between about 1% andabout 2% by weight agar or other non-digestible polysaccharide. Aparticularly preferred seed growth medium contains about 0.05% and about5% by weight of a nitrogen source such as peptone, between about 0.25%and about 2.5% by weight of autolyzed yeast or yeast extract, betweenabout 0.5% and about 5% by weight glucose, and between about 0.05% byweight and about 0.5% by weight of a phosphorus source such as ammoniumphosphate monobasic. Especially economical process operations areafforded by the use of another preferred seed culture which containsbetween about 0.5% and about 5% by weight corn steep liquor, betweenabout 0.25% and about 2.5% autolyzed yeast or yeast extract, betweenabout 0.5% and about 5% by weight glucose and about 0.05% and about 0.5%by weight ammonium phosphate monobasic. Corn steep liquor is aparticularly economical source of proteins, peptides, carbohydrates,organic acids, vitamins, metal ions, trace matters and phosphates. Mashliquors from other grains may be used in place of, or in addition to,corn steep liquor. The pH of the medium is preferably adjusted withinthe range of between about 5.0 and about 7.0, e.g., by addition of analkali metal hydroxide or orthophosphoric acid. Where corn steep liquorserves as the source of nitrogen and carbon, the pH is preferablyadjusted within the range of about 6.2 to about 6.8. The mediumcomprising peptone and glucose is preferably adjusted to a pH betweenabout 5.4 and about 6.2. Among useful growth media for use in seedfermentation:

[0190] 1. Medium #1: 2% peptone, 2% yeast autolised (or yeast extract)and 2% glucose; pH adjusted to 5.8 with 20% NaOH.

[0191] 2. Medium #2: 3% corn steep liquor, 1.5% yeast extract 0.3%ammonium phosphate monobasic and 3% glucose; pH adjusted to 6.5 with 20%NaOH.

[0192] Spores of the microorganism are introduced into this medium froma vial typically containing in the neighborhood of 10⁹ spores per ml. ofsuspension. Optimal productivity of seed generation is realized wheredilution with growth medium at the beginning of a seed culture does notreduce the spore population density below about 10⁷ per ml. Preferably,the spores are cultured in the seed fermentation system until the packedmycelial volume (PMV) in the seed fermenter is at least about 20%,preferably 35% to 45%. Since the cycle in the seed fermentation vessel(or any vessel of a plurality which comprise a seed fermentation train)depends on the initial concentration in that vessel, it may be desirableto provide two or three seed fermentation stages to accelerate theoverall process. However, it is preferable to avoid the use ofsignificantly more than three seed fermenters in series, since activitymay be compromised if seed fermentation is carried through an excessivenumber of stages. The seed culture fermentation is conducted underagitation at a temperature in the range of about 23° to about 37° C.,preferably in range of between about 24° and about 28° C.

[0193] Culture from the seed fermentation system is introduced into aproduction fermenter, together with a production growth medium. In oneembodiment of the invention, non-sterile canrenone or other substrate ofFormula XIII serves as the substrate for the reaction. Preferably, thesubstrate is added to the production fermenter in the form of a 10% to30% by weight slurry in growth medium. To increase the surface areaavailable for 11α-hydroxylation reaction, the particle size of theFormula XIII substrate is reduced by passing the substrate through anoff line micronizer prior to introduction into the fermenter. A sterilenutrient feed stock containing glucose, and a second sterile nutrientsolution containing a yeast derivative such as autolyzed yeast (orequivalent amino acid formulation based on alternative sources such asdistiller's solubles), are also separately introduced. The mediumcomprises an aqueous mixture containing: between about 0.5% and about 5%by weight glucose, between about 0.05% and about 5% by weight of anitrogen source, e.g., peptone, between about 0.05% and about 0.5% byweight of a phosphorus source, e.g., an ammonium or alkali metalphosphate such as dipotassium hydrogen phosphate, between about 0.25%and about 2.5% by weight yeast lysate or extract (or other amino acidsource such as distiller's solubles), between about 1% and about 2% byweight agar or other non-digestible polysaccharide. A particularlypreferred production growth medium contains about 0.05% and about 5% byweight of a nitrogen source such as peptone, between about 0.25% andabout 2.5% by weight of autolyzed yeast or yeast extract, between about0.5% and about 5% by weight glucose, and between about 0.05% and about0.5% by weight of a phosphorus source such as ammonium phosphatemonobasic. Another preferred production medium contains between about0.5% and about 5% by weight corn steep liquor, between about 0.25% andabout 2.5% autolyzed yeast or yeast extract, between about 0.5% andabout 5% by weight glucose and about 0.05% and about 0.5% by weightammonium phosphate monobasic. The pH of the production fermentationmedium is preferably adjusted in the manner described above for the seedfermentation medium, with the same preferred ranges for the pH ofpeptone/glucose based media and corn steep liquor based media,respectively. Useful bioconversion growth media are set forth below:

[0194] 1. Medium #1: 2% peptone, 2% yeast autolised (or yeast extract)and 2% glucose; pH adjusted to 5.8 with 20% NaOH.

[0195] 2. Medium #2: 1% peptone, 1% yeast autolised (or yeast extract)and 2% glucose; pH adjusted to 5.8 with 20% NaOH.

[0196] 3. Medium #3: 0.5% peptone, 0.5% yeast autolised (or yeastextract) and 0.5% glucose; pH adjusted to 5.8 with 20% NaOH.

[0197] 4. Medium #4: 3% corn steep liquor, 1.5% yeast extract 0.3%ammonium phosphate monobasic and 3% glucose; pH adjusted to 6.5 with 20%NaOH.

[0198] 5. Medium #5: 2.55% corn steep liquor, 1.275% yeast extract0.255% ammonium phosphate monobasic and 3% glucose; pH adjusted to 6.5with 20% NaOH.

[0199] 6. Medium #6: 2.1% corn steep liquor, 1.05% yeast extract 0.21%ammonium phosphate monobasic and 3% glucose; pH adjusted to 6.5 with 20%NaOH.

[0200] Non-sterile canrenone and sterile nutrient solutions are chainfed to the production fermenter in five to twenty, preferably ten tofifteen, preferably substantially equal, portions each over theproduction batch cycle. Advantageously, the substrate is initiallyintroduced in an amount sufficient to establish a concentration ofbetween about 0.1% by weight and about 3% by weight, preferably betweenabout 0.5% and about 2% by weight, before inoculation with seedfermentation broth, then added periodically, conveniently every 8 to 24hours, to a cumulative proportion of between about 1% and about 8% byweight. Where additional substrate is added every 8 hour shift, totaladdition may be slightly lower, e.g., 0.25% to 2.5% by weight, than inthe case where substrate is added only on a daily basis. In the latterinstance cumulative canrenone addition may need to be in the range 2% toabout 8% by weight. The supplemental nutrient mixture fed during thefermentation reaction is preferably a concentrate, for example, amixture containing between about 40% and about 60% by weight sterileglucose, and between about 16% and about 32% by weight sterile yeastextract or other sterile source of yeast derivative (or other amino acidsource). Since the substrate fed to the production fermenter of FIG. 1is non-sterile, antibiotics are periodically added to the fermentationbroth to control the growth of undesired organisms. Antibiotics such askanamycin, tetracycline, and cefalexin can be added withoutdisadvantageously affecting growth and bioconversion. Preferably, theseare introduced into the fermentation broth in a concentration, e.g., ofbetween about 0.0004% and about 0.002% based on the total amount of thebroth, comprising, e.g., between about 0.0002% and about 0.0006%kanamicyn sulfate, between about 0.0002% and about 0.006% tetracyclineHCl and/or between about 0.001% and about 0.003% cefalexin, again basedon the total amount of broth.

[0201] Typically, the production fermentation batch cycle is in theneighborhood of 80-160 hours. Thus, portions of each of the Formula XIIIsubstrate and nutrient solutions are typically added every 2 to 10hours, preferably every 4 to 6 hours. Advantageously, an antifoam isalso incorporated in the seed fermentation system, and in the productionfermenter.

[0202] Preferably, in the process of FIG. 1, the inoculum charge to theproduction fermenter is about 0.5 to about 7%, more preferably about 1to about 2%, by volume based on the total mixture in the fermenter, andthe glucose concentration is maintained between about 0.01% and about1.0%, preferably between about 0.025% and about 0.5%, more preferablybetween about 0.05% and about 0.25% by weight with periodic additionsthat are preferably in portions of about 0.05% to about 0.25% by weight,based on the total batch charge. The fermentation temperature isconveniently controlled within a range of about 20° to about 37° C.,preferably about 24° C. to about 28° C., but it may be desirable to stepdown the temperature during the reaction, e.g., in 2° C. increments, tomaintain the packed mycelium volume (PMV) below about 60%, morepreferably below about 50%, and thereby prevent the viscosity of thefermentation broth from interfering with satisfactory mixing. If thebiomass growth extends above the liquid surface, substrate retainedwithin the biomass may be carried out of the reaction zone and becomeunavailable for the hydroxylation reaction. For productivity, it isdesirable to reach a PMV in the range of 30 to 50%, preferably 35% to45%, within the first 24 hours of the fermentation reaction, butthereafter conditions are preferably managed to control further growthwithin the limits stated above. During reaction, the pH of thefermentation medium is controlled at between about 5.0 and about 6.5,preferably between about 5.2 and about 5.8, and the fermenter isagitated at a rate of between about 400 and about 800 rpm. A dissolvedoxygen level of at least about 10% of saturation is achieved by aeratingthe batch at between about 0.2 and about 1.0 vvm, and maintaining thepressure in the head space of the fermenter at between about atmosphericand about 1.0 bar gauge, most preferably in the neighborhood of about0.7 bar gauge. Agitation rate may also been increased as necessary tomaintain minimum dissolved oxygen levels. Advantageously, the dissolvedoxygen is maintained at well above 10%, in fact as high as 50% topromote conversion of substrate. Maintaining the pH in the range of5.5±0.2 is also optimal for bioconversion. Foaming is controlled asnecessary by addition of a common antifoaming agent. After all substratehas been added, reaction is preferably continued until the molar ratioof Formula VIII product to remaining unreacted Formula XIII substrate isat least about 9 to 1. Such conversion may be achieve within the 80-160hour batch cycle indicated above.

[0203] It has been found that high conversions are associated withdepletion of initial nutrient levels below the initial charge level, andby controlling aeration rate and agitation rate to avoid splashing ofsubstrate out of the liquid broth. In the process of FIG. 1, thenutrient level was depleted to and then maintained at no greater thanabout 60%, preferably about 50%, of the initial charge level; while inthe processes of FIGS. 2 and 3, the nutrient level was reduced to andmaintained at no greater than about 80%, preferably about 70%, of theinitial charge level. Aeration rate is preferably no greater than onevvm, more preferably in the range of about 0.5 vvm; while agitation rateis preferably not greater than 600 rpm.

[0204] A particularly preferred process for preparation of a compound ofFormula VIII is illustrated in FIG. 2. Again the preferred microorganismis Aspergillus ochraceus NRRL 405 (ATCC 18500). In this process, growthmedium preferably comprises between about 0.5% and about 5% by weightcorn steep liquor, between about 0.5% and about 5% by weight glucose,between about 0.1% and about 3% by weight yeast extract, and betweenabout 0.05% and about 0.5% by weight ammonium phosphate. However, otherproduction growth media as described herein may also be used. The seedculture is prepared essentially in the manner described for the processof FIG. 1, using any of the seed fermentation media described herein. Asuspension of non-micronized canrenone or other Formula XIII substratein the growth medium is prepared aseptically in a blender, preferably ata relatively high concentration of between about 10% and about 30% byweight substrate. Preferably, aseptic preparation may comprisesterilization or pasteurization of the suspension after mixing. Theentire amount of sterile substrate suspension required for a productionbatch is introduced into the production fermenter at the beginning ofthe batch, or by periodical chain feeding. The particle size of thesubstrate is reduced by wet milling in an on-line shear pump whichtransfers the slurry to the production fermenter, thus obviating theneed for use of an off line micronizer. Where aseptic conditions areachieved by pasteurization rather than sterilization, the extent ofagglomeration may be insignificant, but the use of a shear pump may bedesirable to provide positive control of particle size. Sterile growthmedium and glucose solution are introduced into the production fermenteressentially in the same manner as described above. All feed componentsto the production fermenter are sterilized before introduction, so thatno antibiotics are required.

[0205] Preferably, in operation of the process of FIG. 2, the inoculumis introduced into the production fermenter in a proportion of betweenabout 0.5% and about 7%, the fermentation temperature is between about20° and about 37° C., preferably between about 24° C. and about 28° C.,and the pH is controlled between about 4.4 and about 6.5, preferablybetween about 5.3 and about 5.5, e.g., by introduction of gaseousammonia, aqueous ammonium hydroxide, aqueous alkali metal hydroxide, ororthophosphoric acid. As in the process of FIG. 1, the temperature ispreferably trimmed to control growth of the biomass so that PMV does notexceed 55-60%. The initial glucose charge is preferably between about 1%and about 4% by weight, most preferably 2.5% to 3.5% by weight, but ispreferably allowed to drift below about 1.0% by weight duringfermentation. Supplemental glucose is fed periodically in portions ofbetween about 0.2% and about 1.0% by weight based on the total batchcharge, so as to maintain the glucose concentration in the fermentationzone within a range of between about 0.1% and about 1.5% by weight,preferably between about 0.25% and about 0.5% by weight. Optionally,nitrogen and phosphorus sources may be supplemented along with glucose.However, because the entire canrenone charge is made at the beginning ofthe batch cycle, the requisite supply of nitrogen and phosphorus bearingnutrients can also be introduced at that time, allowing the use of onlya glucose solution for supplementation during the reaction. The rate andnature of agitation is a significant variable. Moderately vigorousagitation promotes mass transfer between the solid substrate and theaqueous phase. However, a low shear impeller should be used to preventdegradation of the myelin of the microorganisms. Optimal agitationvelocity varies within the range of 200 to 800 rpm, depending on culturebroth viscosity, oxygen concentration, and mixing conditions as affectedby vessel, baffle and impeller configuration. Ordinarily, a preferredagitation rate is in the range of 350-600 rpm. Preferably the agitationimpeller provides a downward axially pumping function so as to assist ingood mixing of the fermented biomass. The batch is preferably aerated ata rate of between about 0.3 and about 1.0 vvm, preferably 0.4 to 0.8vvm, and the pressure in the head space of the fermenter is preferablybetween about 0.5 and about 1.0 bar gauge. Temperature, agitation,aeration and back pressure are preferably controlled to maintaindissolved oxygen in the range of at least about 10% by volume during thebioconversion. Total batch cycle is typically between about 100 andabout 140 hours.

[0206] Although the principle of operation for the process of FIG. 2 isbased on early introduction of substantially the entire canrenonecharge, it will be understood that growth of the fermentation broth maybe carried out before the bulk of the canrenone is charged. Optionally,some portion of the canrenone can also be added later in the batch.Generally, however, at least about 75% of the sterile canrenone chargeshould be introduced into the transformation fermenter within 48 hoursafter initiation of fermentation. Moreover, it is desirable to introduceat least about 25% by weight canrenone at the beginning of thefermentation, or at least within the first 24 hours in order to promotegeneration of the bioconversion enzyme(s).

[0207] In a further preferred process as illustrated in FIG. 3, theentire batch charge and nutrient solution are sterilized in theproduction fermentation vessel prior to the introduction of inoculum.The nutrient solutions that may be used, as well as the preferencesamong them, are essentially the same as in the process of FIG. 2. Inthis embodiment of the invention, the shearing action of the agitatorimpeller breaks down the substrate agglomerates that otherwise tend toform upon sterilization. It has been found that the reaction proceedssatisfactorily if the mean particle size of the canrenone is less thanabout 200μ and at least 75% by weight of the particles are smaller than240μ. The use of a suitable impeller, e.g., a disk turbine impeller, atan adequate velocity in the range of 200 to 800 rpm, with a tip speed ofat least about 400 cm/sec., has been found to provide a shear ratesufficient to maintain such particle size characteristics despite theagglomeration that tends to occur upon sterilization within theproduction fermenter. The remaining operation of the process of FIG. 3is essentially the same as the process of FIG. 2. The processes of FIGS.2 and 3 offer several distinct advantages over the process of FIG. 1. Aparticular advantage is the amenability to use of a low cost nutrientbase such as corn steep liquor. But further advantages are realized ineliminating the need of antibiotics, simplifying feeding procedures, andallowing for batch sterilization of canrenone or other Formula XIIIsubstrate. Another particular advantage is the ability to use a simpleglucose solution rather than a complex nutrient solution forsupplementation during the reaction cycle.

[0208] In processes depicted in FIGS. 1 to 3, the product of Fig. VIIIis a crystalline solid which, together with the biomass, may beseparated from the reaction broth by filtration or low speedcentrifugation. Alternatively, the product can be extracted from theentire reaction broth with organic solvents. Product of Formula VIII isrecovered by solvent extraction. For maximum recovery, both the liquidphase filtrate and the biomass filter or centrifuge cake are treatedwith extraction solvent, but usually ≧95% of the product is associatedwith the biomass. Typically, hydrocarbon, ester, chlorinatedhydrocarbon, and ketone solvents may be used for extraction. A preferredsolvent is a ethyl acetate. Other typically suitable solvents includetoluene and methyl isobutyl ketone. For extraction from the liquidphase, it may be convenient to use a volume of solvent approximatelyequal to the volume of reaction solution which it contacts. To recoverproduct the from the biomass, the latter is suspended in the solvent,preferably in large excess relative to the initial charge of substrate,e.g., 50 to 100 ml. solvent per gram of initial canrenone charge, andthe resulting suspension preferably refluxed for a period of 20 minutesto several hours to assure transfer of product to the solvent phase fromrecesses and pores of the biomass. Thereafter, the biomass is removed byfiltration or centrifugation, and the filter cake preferably washed withboth fresh solvent and deionized water. Aqueous and solvent washes arethen combined and the phases allowed to separate. Formula VIII productis recovered by crystallization from the solution. To maximize yield,the mycelium is contacted twice with fresh solvent. After settling toallow complete separation of the aqueous phase, product is recoveredfrom the solvent phase. Most preferably, the solvent is removed undervacuum until crystallization begins, then the concentrated extract iscooled to a temperature of 0° to 20° C., preferably about 10° to about15° C. for a time sufficient for crystal precipitation and growth,typically 8 to 12 hours.

[0209] The processes of FIG. 2, and especially that of FIG. 3, areparticularly preferred. These processes operate at low viscosity, andare amenable to close control of process parameters such as pH,temperature and dissolved oxygen. Moreover, sterile conditions arereadily preserved without resort to antibiotics.

[0210] The bioconversion process is exothermic, so that heat should beremoved, using a jacketed fermenter or a cooling coil within theproduction fermenter. Alternatively, the reaction broth may becirculated through an external heat exchanger. Dissolved oxygen ispreferably maintained at a level of at least about 5%, preferably atleast about 10%, by volume, sufficient to provide energy for thereaction and assure conversion of the glucose to CO₂ and H₂O, byregulating the rate of air introduced into the reactor in response tomeasurement of oxygen potential in the broth. The pH is preferablycontrolled at between about 4.5 and about 6.5.

[0211] In each of the alternative processes for 11-hydroxylation of thesubstrate of Formula XIII, productivity is limited by mass transfer fromthe solid substrate to the aqueous phase, or the phase interface, wherereaction is understood to occur. As indicated above, productivity is notsignificantly limited by mass transfer rates so long as the particlemean particle size of the substrate is reduced to less than about 300μ,and at least 75% by weight of the particles are smaller than 240μ.However, productivity of these processes may be further enhanced incertain alternative embodiments which provide a substantial charge ofcanrenone or other Formula XIII substrate to the production fermenter inan organic solvent. According to one option, the substrate is dissolvedin a water-immiscible solvent and mixed with the aqueous growth mediuminoculum and a surfactant. Useful water-immiscible solvents inlcude, forexample, DMF, DMSO, C₆-C₁₂ fatty acids, C₆-C₁₂ n-alkanes, vegetableoils, sorbitans, and aqueous surfactant solutions. Agitation of thischarge generates an emulsion reaction system having an extendedinterfacial area for mass transfer of substrate from the organic liquidphase to the reaction sites.

[0212] A second option is to initially dissolve the substrate in a watermiscible solvent such as acetone, methylethyl ketone, methanol, ethanol,or glycerol in a concentration substantially greater than its solubilityin water. By preparing the initial substrate solution at elevatedtemperature, solubility is increased, thereby further increasing theamount of solution form substrate introduced into the reactor andultimately enhancing the reactor payload. The warm substrate solution ischarged to the production fermentation reactor along with the relativelycool aqueous charge comprising growth medium and inoculum. When thesubstrate solution is mixed with the aqueous medium, precipitation ofthe substrate occurs. However, under conditions of substantialsupersaturation and moderately vigorous agitation, nucleation is favoredover crystal growth, and very fine particles of high surface area areformed. The high surface area promotes mass transfer between the liquidphase and the solid substrate. Moreover, the equilibrium concentrationof substrate in the aqueous liquid phase is also enhanced in thepresence of a water-miscible solvent. Accordingly, productivity ispromoted.

[0213] Although the microorganism may not necessarily tolerate a highconcentration of organic solvent in the aqueous phase, a concentrationof ethanol, e.g., in the range of about 3% to about 5% by weight, can beused to advantage.

[0214] A third option is to solubilize the substrate in an aqueouscyclodextrin solution. Illustrative cyclodextrins includehydroxypropyl-β-cyclodextrin and methyl-β-cyclodextrin. The molar ratioof substrate:cyclodextrin can be about 1:1 to about 1:1.5,substrate:cyclodextrin. The substrate:cyclodextrin mixture can then beadded aseptically to the bioconversion reactor.

[0215] 11α-Hydroxycanrenone and other products of the 11α-hydroxylationprocess (Formulae VIII and VIIIA) are novel compounds, which may beisolated by filtering the reaction medium, and extracting the productfrom the biomass collected on the filtration medium. Conventionalorganic solvents, e.g., ethyl acetate, acetone, toluene, chlorinatedhydrocarbons, and methyl isobutyl ketone may be used for the extraction.The product of Formula VIII may then be recrystallized from an organicsolvent of the same type. The compounds of Formula VIII have substantialvalue as intermediates for the preparation of compounds of Formula I,and especially of Formula IA.

[0216] Preferably, the compounds of Formula VIII correspond to FormulaVIIIA in which —A—A— and —B—B— are —CH₂—CH₂—, R³ is hydrogen, loweralkyl or lower alkoxy, and R⁸ and R⁹ together constitute the20-spiroxane ring:

[0217] Further in accordance with the process of scheme 1, the compoundof Formula VIII is reacted under alkaline conditions with a source ofcyanide ion to produce an enamine compound of Formula VII

[0218] wherein —A—A—, R³, —B—B—, R⁸ and R⁹ are as defined above. Wherethe substrate corresponds to Formula VIIIA, the product is of FormulaVIIA

[0219] wherein —A—A—, —B—B—, R³, Y¹, Y², and X are as defined in FormulaXIII.

[0220] Cyanidation of the 11α-hydroxyl substrate of Formula VIII may becarried out by reacting it with a cyanide ion source such as a ketonecyanohydrin, most preferably acetone cyanohydrin, in the presence of abase and a alkali metal salt, most preferably LiCl. Alternatively,cyanidation can be effected without a cyanohydrin by using an alkalimetal cyanide in the presence of an acid.

[0221] In the ketone cyanohydrin process, the reaction is conducted insolution, preferably using an aprotic polar solvent such asdimethylformamide or dimethyl sulfoxide. Formation of the enaminerequires at least two moles of cyanide ion source per mole of substrate,and preferably a slight excess of the cyanide source is used. The baseis preferably a nitrogenous base such as a dialkylamine, trialkylamine,alkanolamine, pyridine or the like. However, inorganic bases such asalkali metal carbonates or alkali metal hydroxides can also be used.Preferably, the substrate of Formula VIII is initially present in aproportion of between about 20 and about 50% by weight and the base ispresent in a proportion of between 0.5 to two equivalents per equivalentof substrate. The temperature of the reaction is not critical, butproductivity is enhanced by operation at elevated temperature. Thus, forexample, where triethylamine is used as the base, the reaction isadvantageously conducted in the range of about 80° C. to about 90° C. Atsuch temperatures, the reaction proceeds to completion in about 5 toabout 20 hours. When diisopropylethyl amine is used as the base and thereaction is conducted at 105° C., the reaction is completed at 8 hours.At the end of the reaction period, the solvent is removed under vacuumand the residual oil dissolved in water and neutralized to pH 7 withdilute acid, preferably hydrochloric. The product precipitates from thissolution, and is thereafter washed with distilled water and air dried.Liberated HCN may be stripped with an inert gas and quenched in analkaline solution. The dried precipitate is taken up in chloroform orother suitable solvent, then extracted with concentrated acid, e.g., 6NHCl. The extract is neutralized to pH 7 by addition of an inorganicbase, preferably an alkali metal hydroxide, and cooled to a temperaturein the range of 0° C. The resulting precipitate is washed and dried,then recrystallized from a suitable solvent, e.g., acetone, to produce aproduct of Formula VII suitable for use in the next step of the process.

[0222] Alternatively, the reaction may be conducted in a aqueous solventsystem comprising water-miscible organic solvent such as methanol or ina biphasic system comprising water and an organic solvent such as ethylacetate. In this alternative, product may be recovered by diluting thereaction solution with water, and thereafter extracting the productusing an organic solvent such as methylene chloride or chloroform, andthen back extracting from the organic extract using concentrated mineralacid, e.g., 2N HCl. See U.S. Pat. No. 3,200,113.

[0223] According to a still further alternative, the reaction may beconducted in a water-miscible solvent such as dimethylformamide,dimethylacetamide, N-methyl, pyrolidone or dimethyl sulfoxide, afterwhich the reaction product solution is diluted with water and renderedalkaline, e.g., by addition of an alkali metal carbonate, then cooled to0° to 10° C., thereby causing the product to precipitate. Preferably,the system is quenched with an alkali metal hypohalite or other reagenteffective to prevent evolution of cyanide. After filtration and washingwith water, the precipitated product is suitable for use in the nextstep of the process.

[0224] According to a still further alternative, the enamine product ofFormula VII may be produced by reaction of a substrate of Formula VIIIin the presence of a proton source, with an excess of alkali metalcyanide, preferably NaCN, in an aqueous solvent comprising an aproticwater-miscible polar solvent such as dimethylformamide ordimethylacetamide. The proton source is preferably a mineral acid or C₁to C₅ carboxylic acid, sulfuric acid being particularly preferred.Anomalously, no discrete proton source need be added where thecyanidation reagent is commercial LiCN in DMF.

[0225] Cyanide ion is preferably charged to the reactor in a proportionof between about 2.05 and about 5 molar equivalents per equivalent ofsubstrate. The mineral acid or other proton source is believed topromote addition of HCN across the 4,5 and 6,7 double bonds, and ispreferably present in a proportion of at least one mole equivalent permole equivalent substrate; but the reaction system should remain basicby maintaining an excess of alkali metal cyanide over acid present.Reaction is preferably carried out at a temperature of at least about75° C., typically 60° C. to 100° C., for a period of about 1 to about 8hours, preferably about 1.5 to about 3 hours. At the end of the reactionperiod, the reaction mixture is cooled, preferably to about roomtemperature; and the product enamine is precipitated by acidifying thereaction mixture and mixing it with cold water, preferably at about icebath temperature. Acidification is believed to close the 17-lactone,which tends to open under the basic conditions prevailing in thecyanidation. The reaction mixture is conveniently acidified using thesame acid that is present during the reaction, preferably sulfuric acid.Water is preferably added in a proportion of between about 10 and about50 mole equivalents per mole of product.

[0226] The compounds of Formula VII are novel compounds and havesubstantial value as intermediates for the preparation of compounds ofFormula I, and especially of Formula IA. Preferably, the compounds ofFormula VII correspond to Formula VIIA in which —A—A— and —B—B— are—CH₂—CH₂—, R³ is hydrogen, lower alkyl or lower alkoxy, and R⁸ and R⁹together constitute the 20-spiroxane ring:

[0227] Most preferably the compound of Formula VII is5′R(5′α),7′β-20′-Aminohexadecahydro-11′β-hydroxy-10′α,13′α-dimethyl-3′,5-dioxospiro[furan-2(3H),17′β(5′H)-[7,4]metheno[4H]cyclopenta[a]phenanthrene]-5′-carbonitrile.

[0228] In the next step of the Scheme 1 synthesis, the enamine ofFormula VII is hydrolyzed to produce a diketone compound of formula VI

[0229] where —A—A—, R³, —B—B—, R⁸ and R⁹ are as defined in Formula VIII.Any aqueous organic or mineral acid can be used for the hydrolysis.Hydrochloric acid is preferred. To enhance productivity, awater-miscible organic solvent, such as a lower alkanol, is preferablyused as a cosolvent. The acid should be present in proportion of atleast one equivalent per equivalent of Formula VII substrate. In anaqueous system, the enamine substrate VII can be substantially convertedto the diketone of Formula VII in a period of about 5 hours at about 80°C. Operation at elevated temperature increases productivity, buttemperature is not critical. Suitable temperatures are selected based onthe volatility of the solvent system and acid.

[0230] Preferably, the enamine substrate of Formula VII corresponds toFormula VIIA

[0231] and the diketone product corresponds to Formula VIA

[0232] in each of which —A—A—, —B—B—, Y¹, Y², and X are as defined inFormula VIIIA.

[0233] At the end of the reaction period, the solution is cooled to 0°and 25° C. to crystallize the product. The product crystals may berecrystallized from a suitable solvent such as isopropanol or methanolto produce a product of Formula VI suitable for use in the next step ofthe process; but recrystallization is usually not necessary. Theproducts of Formula VI are novel compounds which have substantial valueas intermediates for the preparation of compounds of Formula I, andespecially of Formula IA. Preferably, the compounds of Formula VIcorrespond to Formula VIA in which —A—A— and —B—B— are —CH₂—CH₂—, R³ ishydrogen, lower alkyl or lower alkoxy, and R⁸ and R⁹ together constitutethe 20-spiroxane ring:

[0234] Most preferably, the compound of Formula VI is4′S(4′α),7′α-Hexadecahydro-11′α-hydroxy-10′β,13′β-dimethyl-3′,5,20′-trioxospiro[furan-2(3H),17′β-[4,7]methano[17H]cyclopenta[a]phenanthrene]-5′β(2′H)-carbonitrile.

[0235] In a particularly preferred embodiment of the invention, theproduct enamine of Formula VII is produced from the compound of FormulaVIII in the manner described above, and converted in situ to thediketone of Formula VI. In this embodiment of the invention, a formulaVIII substrate is reacted with an excess of alkali metal cyanide in anaqueous solvent containing a proton source, or optionally an excess ofketone cyanohydrin in the presence of a base and LiCl, as describedhereinabove. However, instead of cooling the reaction mixture,acidifying, and adding water in proportions calculated to causeprecipitation of the enamine, substantial cooling of the reactionmixture is preferably avoided. Water and an acid, preferably a mineralacid such as sulfuric, are indeed added to mixture at the end of thecyanidation reaction, and the proportion of acid added is sufficient toneutralize excess alkali metal cyanide, which ordinarily requiresintroduction of at least one molar equivalent acid per mole of FormulaVIII substrate, preferably between about 2 and about 5 mole equivalentsper equivalent substrate. However, the temperature is maintained at highenough, and the dilution greater enough, so that substantialprecipitation is avoided and hydrolysis of the enamine to the diketoneis allowed to proceed in the liquid phase. Thus, the process proceedswith minimum interruption and high productivity. Hydrolysis ispreferably conducted at a temperature of at least 80° C., morepreferably in the range of about 90° C. to about 100° C., for a periodof typically about 1 to about 10 hours, more preferably about 2 to about5 hours. Then the reaction mixture is cooled, preferably to atemperature of between about 0° C. and about 15° C., advantageously inan ice bath to about 5° C. to about 10° C., for precipitation of theproduct diketone of Formula VI. The solid product may be recovered, asby filtration, and impurities removed by washing with water.

[0236] In the next step of the Scheme 1 synthesis, the diketone compoundof Formula VI is reacted with a metal alkoxide to open up the ketonebridge between the 4 and 7 positions, cleave the bond between thecarbonyl group and the 4-carbon, and form an α-orientedalkanoyloxycarbonyl substituent at the 7 position and eliminatingcyanide at the 5-carbon. The product of this reaction is a hydroxyestercompound corresponding to Formula V

[0237] where —A—A—, R³, —B—B—, R⁸ and R⁹ are as defined in Formula VIII,and R¹ is lower alkoxycarbonyl or hydroxycarbonyl. The metal alkoxideused in the reaction corresponds to the formula R¹⁰OM where M is alkalimetal and R¹⁰ corresponds to the alkoxy substituent of R¹. Yields ofthis reaction are most satisfactory when the metal alkoxide is K or Namethoxide, but other lower alkoxides can be used. A K alkoxide isparticularly preferred. Phenoxides, other aryloxides may also be used,as well as arylsulfides. The reaction is conveniently carried out in thepresence of an alcohol corresponding to the formula R¹⁰OH where R¹⁰ isas defined above. Other conventional solvents may be used. Preferably,the Formula VI substrate is present in a proportion of between about 2%and about 12% by weight, more preferably at least about 6% by weight andR¹⁰OM is present in a proportion of between about 0.5 and about 4 molesper mole of substrate. Temperature is not critical but elevatedtemperature enhances productivity. Reaction time is typically betweenabout 4 and about 24 hours, preferably about 4 to 16 hours.Conveniently, the reaction is carried out at atmospheric refluxtemperature depending on the solvent used.

[0238] In the conversion of the diketone of Formula VI to thehydroxyester of Formula VI, by-product cyanide ion can react with theproduct to form 5-cyanoester. Because the equilibrium is more favorableat low concentrations, the reaction is preferably run at rather highdilution, e.g., as high as 40:1 for reaction with Na methoxide. It hasbeen found that significantly higher productivity can be realized by useof K methoxide rather than Na methoxide, because a dilution in the rangeof about 20:1 is generally sufficient to minimize the extent of reversecyanidation where K methoxide is the reagent.

[0239] In accordance with the invention, it has been further discoveredthat the reverse cyanidation reaction may be inhibited by takingappropriate chemical or physical measures to remove by-product cyanideion from the reaction zone. Thus, in a further embodiment of theinvention, the reaction of the diketone with alkali metal alkoxide maybe carried out in the presence of an precipitating agent for cyanide ionsuch as, for example, a salt comprising a cation which forms aninsoluble cyanide compound. Such salts may, for example, include zinciodide, ferric sulfate, or essentially any halide, sulfate or other saltof an alkaline earth or transition metal that is more soluble than thecorresponding cyanide. If zinc iodide is present in proportions in therange of about one equivalent per equivalent diketone substrate, it hasbeen observed that the productivity of the reaction is increasedsubstantially as compared to the process as conducted in the absence ofan alkali metal halide.

[0240] Even where a precipitating agent is used for removal of cyanideion, it remains preferable to run at fairly high dilution, but by use ofa precipitating agent the solvent to diketone substrate molar ratio maybe reduced significantly compared to reactions in the absence of suchagent. Recovery of the hydroxyester of Formula V can be carried outaccording to either the extractive or non-extractive proceduresdescribed below.

[0241] Preferably, the diketone substrate of Formula VI corresponds toFormula VIA

[0242] and the hydroxyester product corresponds to Formula VA

[0243] in each of which —A—A—, —B—B—, Y¹, Y², and X are as defined inFormula XIII and R¹ is as defined in Formula V.

[0244] The products of Formula V are novel compounds which havesubstantial value as intermediates for the preparation of compounds ofFormula I, and especially of Formula IA. Preferably, the compounds ofFormula V correspond to Formula VA in which —A—A— and —B—B— are—CH₂—CH₂—, R³ is hydrogen, lower alkyl or lower alkoxy, and R⁸ and R⁹together constitute the 20-spiroxane ring:

[0245] Most preferably, the compound of Formula V is Methyl Hydrogen11α,17α-Dihydroxy-3-oxopregn-4-ene-7α,21-dicarboxylate, γ-Lactone.

[0246] The compound of Formula V may be isolated by acidifying thereaction solution, e.g., with concentrated HCl, cooling to ambienttemperature, and extracting the product with an organic solvent such asmethylene chloride or ethyl acetate. The extract is washed with anaqueous alkaline wash solution, dried and filtered, after which thesolvent is removed. Alternatively, the reaction solution containing theproduct of Formula V may be quenched with concentrated acid. The productsolution is concentrated, cooled to 0° to 25° C. and the product solidis isolated by filtration.

[0247] According to a preferred mode of recovery of the product ofFormula V, methanol and HCN are removed by distillation after theconclusion of the reaction period, with water and acid being addedbefore or during the distillation. Addition of water before thedistillation simplifies operations, but progressive addition during thedistillation allows the volume in the still to be maintainedsubstantially constant. Product of Formula V crystallizes from the stillbottoms as the distillation proceeds. This mode of recovery provides ahigh quality crystalline product without extraction operations.

[0248] In accordance with a further alternative, the reaction solutioncontaining the product of Formula V may be quenched with mineral acid,e.g., 4N HCl, after which the solvent is removed by distillation.Removal of the solvent is also effective for removing residual HCN fromthe reaction product. It has been found that multiple solventextractions for purification of the compound of Formula V are notnecessary where the compound of Formula V serves as an intermediate in aprocess for the preparation of epoxymexrenone, as described herein. Infact, such extractions can often be entirely eliminated. Where solventextraction is used for product purification, it is desirable tosupplement the solvent washes with brine and caustic washes. But wherethe solvent extractions are eliminated, the brine and caustic washes aretoo. Eliminating the extractions and washes significantly enhances theproductivity of the process, without sacrificing yield or productquality, and also eliminates the need for drying of the washed solutionwith a dessicant such as sodium sulfate. The crude11α-hydroxy-7α-alkanoyloxycarbonyl product is taken up again in thesolvent for the next reaction step of the process, which is theconversion of the 11-hydroxy group to a good leaving group at the 11position thereby producing a compound of Formula IV:

[0249] where —A—A—, R³, —B—B—, R⁸ and R⁹ are as defined in Formula VIII,R¹ is as defined in Formula V, and R² is lower arylsulfonyloxy,alkylsulfonyloxy, acyloxy or halide. Preferably, the 11α-hydroxyl isesterified by reaction with a lower alkylsulfonyl halide, an acyl halideor an acid anhydride which is added to the solution containing theintermediate product of Formula V. Lower alkylsulfonyl halides, andespecially methanesulfonyl chloride, are preferred. Alternatively, the11-α hydroxy group could be converted to a halide by reaction of asuitable reagent such as thionyl bromide, thionyl chloride, sulfurylchloride or oxalyl chloride. Other reagents for forming 11α-sulfonicacid esters include tosyl chloride, benzenesulfonyl chloride andtrifluoromethanesulfonic anhydride. The reaction is conducted in asolvent containing a hydrogen halide scavenger such as triethylamine orpyridine. Inorganic bases such as K or Na carbonate can also be used.The initial concentration of the hydroxyester of Formula V is preferablybetween about 5% and about 50% by weight. The esterification reagent ispreferably present in slight excess. Methylene chloride is aparticularly suitable solvent for the reaction, but other solvents suchas dichloroethane, pyridine, chloroform, methyl ethyl ketone,dimethoxyethane, methyl isobutyl ketone, acetone, other ketones, ethers,acetonitrile, toluene, and tetrahydrofuran can also be employed. Thereaction temperature is governed primarily by the volatility of thesolvent. In methylene chloride, the reaction temperature is preferablyin the range of between about −10° C. and about 10° C.

[0250] Preferably, the hydroxyester substrate of Formula V correspondsto Formula VA

[0251] and the product corresponds to Formula IVA

[0252] in each of which —A—A—, —B—B—, Y¹, Y², and X are as defined inFormula XIII, R¹ is lower alkanoyloxycarbonyl or hydroxycarbonyl, and R²is as defined in Formula IV.

[0253] The products of Formula IV are novel compounds which havesubstantial value as intermediates for the preparation of compounds ofFormula I, and especially of Formula IA. Preferably, the compounds ofFormula IV correspond to Formula VA in which —A—A— and —B—B— are—CH₂—CH₂—, R³ is hydrogen, lower alkyl or lower alkoxy, and R⁸ and R⁹together constitute the 20-spiroxane ring:

[0254] Most preferably, the compound of Formula IV is Methyl Hydrogen17α-Hydroxy-11α-(methylsulfonyl)oxy-3-oxopregn-

[0255]4-ene-7α,21-dicarboxylate, γ-Lactone.

[0256] If desired, the compound of Formula IV may be isolated by removalof the solvent. Preferably, the reaction solution is first washed withan aqueous alkaline wash solution, e.g., 0.5-2N NaoH, followed by anacid wash, e.g., 0.5-2N HCl. After removal of the reaction solvent, theproduct is recrystallized, e.g., by taking the product up in methylenechloride and then adding another solvent such as ethyl ether whichlowers the solubility of the product of Formula IV, causing it toprecipitate in crystalline form.

[0257] In the recovery of the product of Formula IV, or in preparationof the reaction solution for conversion of the Formula IV intermediateto the intermediate of Formula II as is further described hereinbelow,all extractions and/or washing steps may be dispensed with if thesolution is instead treated with ion exchange resins for removal ofacidic and basic impurities. The solution is treated first with an anionexchange resin, then with a cation exchange resin. Alternatively, thereaction solution may first be treated with inorganic adsorbents such asbasic alumina or basic silica, followed by a dilute acid wash. Basicsilica or basic alumina may typically be mixed with the reactionsolution in a proportion of between about 5 and about 50 g per kg ofproduct, preferably between about 15 and about 20 g per kg product.Whether ion exchange resins or inorganic adsorbents are used, thetreatment can be carried out by simply slurrying the resin or inorganicadsorbent with the reaction solution under agitation at ambienttemperature, then removing the resin or inorganic adsorbent byfiltration.

[0258] In an alternative and preferred embodiment of the invention, theproduct compound of Formula IV is recovered in crude form as aconcentrated solution by removal of a portion of the solvent. Thisconcentrated solution is used directly in the following step of theprocess, which is removal of the 11α-leaving group from the compound ofFormula IV, thereby producing an enester of Formula II:

[0259] where —A—A—, R³, —B—B—, R⁸ and R⁹ are as defined in Formula VIII,and R¹ is as defined in Formula V. For purposes of this reaction, the R²substituent of the compound of Formula IV may be any leaving group theabstraction of which is effective for generating a double bond betweenthe 9- and 11-carbons. Preferably, the leaving group is a loweralkylsulfonyloxy or acyloxy substituent which is removed by reactionwith an acid and an alkali metal salt. Mineral acids can be used, butlower alkanoic acids are preferred. Advantageously, the reagent for thereaction further includes an alkali metal salt of the alkanoic acidutilized. It is particularly preferred that the leaving group comprisemesyloxy and the reagent for the reaction comprise formic acid or aceticacid and an alkali metal salt of one of these acids or another loweralkanoic acid. Where the leaving group is mesyloxy and the removalreagent is formic acid and potassium formate a relatively high ratio of9,11 to 11,12-olefin is observed. If free water is present duringremoval of the leaving group, impurities tend to be formed, particularlya 7,9-lactone

[0260] which is difficult to remove from the final product. Hence,acetic anhydride or other drying agent is used to remove the waterpresent in formic acid. The free water content of the reaction mixturebefore reaction should be maintained at a level below about 0.5%,preferably below about 0.1% by weight, as measured by Karl Fischeranalysis for water, based on total reaction solution. Although it ispreferred that the reaction mixture be kept as dry as practicable,satisfactory results have been realized with 0.3% by weight water.Preferably, the reaction charge mixture contains between about 4% andabout 50% by weight of the substrate of Formula IV in the alkanoic acid.Between about 4% and about 20% by weight of the alkali metal salt of theacid is preferably included. Where acetic anhydride is used as thedrying agent, it is preferably present in a proportion of between about0.05 moles and about 0.2 moles per mole of alkanoic acid.

[0261] It has been found that proportions of by-product 7,9-lactone and11,12-olefin in the reaction mixture is relatively low where theelimination reagent comprises a combination of trifluoroacetic acid,trifluoroacetic anhydride and potassium acetate as the reagent forelimination of the leaving group and formation of the enester(9,11-olefin). Trifluoroacetic anhydride serves as the drying agent, andshould be present in a proportion of at least about 3% by weight, morepreferably at least about 15% by weight, most preferably about 20% byweight, based on the trifluoroacetic acid eliminating reagent.

[0262] Alternatively, the 11α-leaving groups from the compound ofFormula IV, may be eliminated to produce an enester of Formula II byheating a solution of Formula IV in an organic solvent such as DMSO, DMFor DMA.

[0263] Further in accordance with the invention, the compound of FormulaIV is reacted initially with an alkenyl alkanoate such as isopropenylacetate in the presence of an acid such as toluene sulfonic acid or ananhydrous mineral acid such as sulfuric acid to form the 3-enol ester:

[0264] of the compound of Formula IV. Alternatively, the 3 enol estercan be formed by treatment with an acid anhydrides and base such asacetic acid and sodium acetate. Further alternatives include treatmentwith ketene in the presence of an acid to produce IV(Z). Theintermediate of Formula IV(Z) is thereafter reacted with an alkali metalformate or acetate in the presence of formic or acetic acid to producethe Δ-9,11 enol acetate of Formula IV(Y):

[0265] which can then be converted to the enester of Formula II in anorganic solvent, preferably an alcohol such as methanol, by eitherthermal decomposition of the enol acetate or reaction thereof with analkali metal alkoxide. The elimination reaction is highly selective tothe enester of Formula II in preference to the 11,12-olefin and7,9-lactone, and this selectivity is preserved through conversion of theenol acetate to the enone.

[0266] Preferably, the substrate of Formula IV corresponds to FormulaIVA

[0267] in each of which —A—A—, —B—B—, Y¹, Y², and X are as defined inFormula XIII and R¹ is as defined in Formula V.

[0268] If desired, the compound of Formula II may be isolated byremoving the solvent, taking up the solid product in cold water, andextracting with an organic solvent, such as ethyl acetate. Afterappropriate washing and drying steps, the product is recovered byremoving the extraction solvent. The enester is then dissolved in asolvent appropriate for the conversion to the product of Formula I.Alternatively, the enester can be isolated by adding water to theconcentrated product solution and filtering the solid product, therebypreferentially removing the 7,9-lactone. Conversion of the substrate ofFormula II to the product of Formula IA may be conducted in the mannerdescribed in U.S. Pat. No. 4,559,332 which is expressly incorporatedherein by reference, or more preferably by the novel reaction using ahaloacetamide promoter as described below.

[0269] In another embodiment of the invention, the hydroxyester ofFormula V may be converted to the enester of Formula II withoutisolation of the intermediate compound of Formula IV. In this method,the hydroxyester is taken up in a an organic solvent, such as methylenechloride; and either an acylating agent, e.g., methanesulfonyl chloride,or halogenating reagent, e.g., sulfuryl chloride, is added to thesolution. The mixture is agitated and, where halogenation is involved,an HCl scavenger such as imidazole is added. Mixing of base with thesolution is highly exothermic, and should therefore be conducted at acontrolled rate with full cooling. After the base addition, theresulting mixture is warmed to moderate temperature, e.g., 0° C. to roomtemperature or slightly above, and reacted for a period of typically 1to 4 hours. After reaction is complete, the solvent is stripped,preferably under high vacuum (e.g., 24″ to 28″ Hg) conditions at −10° to+15° C., more preferably about 0° to about 5° C., to concentrate thesolution and remove excess base. The substrate is then redissolved in anorganic solvent, preferably a halogenated solvent such as methylenechloride for conversion to the enester.

[0270] The leaving group elimination reagent is preferably prepared bymixing an organic acid, an organic acid salt and a drying agent,preferably formic acid, alkali metal formate and acetic anhydride,respectively, in a dry reactor. Addition of acetic anhydride isexothermic and results in release of CO, so the addition rate must becontrolled accordingly. To promote the removal of water, the temperatureof this reaction is preferably maintained in the range of 60° to 90° C.,most preferably about 65° to about 75° C. This reagent is then added tothe product solution of the compound of Formula IV to effect theelimination reaction. After 4-8 hours, the reaction mixture ispreferably heated to a temperature of at least about 85° C., but notabove about 95° C. until all volatile distillate has been removed, andthen for an additional period to complete the reaction, typically about1 to 4 hours. The reaction mixture is cooled, and after recovery bystandard extraction techniques, the enester may be recovered as desiredby evaporating the solvent.

[0271] It has further been found that the enester of Formula II may berecovered from the reaction solution by an alternative procedure whichavoids the need for extraction steps following the elimination reaction,thereby providing savings in cost, improvement in yield and/orimprovement in productivity. In this process, the enester product isprecipitated by dilution of the reaction mixture with water afterremoval of formic acid. The product is then isolated by filtration. Noextractions are required.

[0272] According to a further alternative for conversion of thehydroxyester of Formula V to the enester of Formula II without isolationof the compound of Formula IV, the 11α-hydroxy group of the Formula Vhydroxyester is replaced by halogen, and the Formula II enester is thenformed in situ by thermal dehydro halogenation. Replacement of thehydroxy group by halogen is effected by reaction with sulfuryl halide,preferably sulfuryl chloride, in the cold in the presence of a hydrogenhalide scavenger such as imidazole. The hydroxyester is dissolved in asolvent such as tetrahydrofuran and cooled to 0° C. to −70° C. Thesulfuryl halide is added and the reaction mixture is warmed to moderatetemperature, e.g., room temperature, for a time sufficient to completethe elimination reaction, typically 1 to 4 hours. The process of thisembodiment not only combines two steps into one, but eliminates the useof: a halogenated reaction solvent; an acid (such as acetic); and adrying reagent (acetic anhydride or sodium sulfate). Moreover, thereaction does not require refluxing conditions, and avoids thegeneration of by-product CO which results when acetic acid is used as adrying reagent.

[0273] In accordance with a particularly preferred embodiment of theinvention, the diketone compound of Formula VI can be converted toepoxymexrenone or other compound of Formula I without isolating anyintermediate in purified form. In accordance with this preferredprocess, the reaction solution containing the hydroxyester is quenchedwith a strong acid solution, cooled to ambient temperature and thenextracted with an appropriate extraction solvent. Advantageously, anaqueous solution of inorganic salt, e.g., 10% by weight saline solution,is added to the reaction mixture prior to the extraction. The extract iswashed and dried by azeotropic distillation for removal of the methanolsolvent remaining from the ketone cleavage reaction.

[0274] The resulting concentrated solution containing between about 5%and about 50% by weight compound of Formula V is then contacted in thecold with an acylating or alkylsulfonylating reagent to form thesulfonic ester or dicarboxylic acid ester. After the alkylsulfonation orcarboxylation reaction is complete the reaction solution is passed overan acidic and then a basic exchange resin column for the removal ofbasic and acidic impurities. After each pass, the column is washed withan appropriate solvent, e.g., methylene chloride, for the recovery ofresidual sulfonic or dicarboxylic ester therefrom. The combined eluateand wash fractions are combined and reduced, preferably under vacuum, toproduce a concentrated solution containing the sulfonic ester ordicarboxylic ester of Formula IV. This concentrated solution is thencontacted with a dry reagent comprising an agent effect for removal ofthe 11α-ester leaving group and abstraction of hydrogen to form a 9,11double bond. Preferably, the reagent for removal of the leaving groupcomprises the formic acid/alkali metal formate/acetic anhydride dryreagent solution described above. After reaction is complete, thereaction mixture is cooled and formic acid and/or other volatilecomponents are removed under vacuum. The residue is cooled to ambienttemperature, subjected to appropriate washing steps, and then dried togive a concentrated solution containing the enester of Formula II. Thisenester may then be converted to epoxymexrenone or other compound ofFormula I using the method described herein, or in U.S. Pat. No.4,559,332.

[0275] In an especially preferred embodiment of the invention, thesolvent is removed from the reaction solution under vacuum, and theproduct of Formula IV is partitioned between water and an appropriateorganic solvent, e.g., ethyl acetate. The aqueous layer is then backextracted with the organic solvent, and the back extract washed with analkaline solution, preferably a solution of an alkali metal hydroxidecontaining an alkali metal halide. The organic phase is concentrated,preferably under vacuum, to yield the enester product of Formula II. Theproduct of Formula II may then be taken up in an organic solvent, e.g.,methylene chloride, and further reacted in the manner described in the'332 patent to produce the product of Formula I.

[0276] Where trihaloacetonitrile is used in the epoxidation reaction, ithas been found that the selection of solvent is important, withhalogenated solvents being highly preferred, and methylene chloridebeing especially preferred. Solvents such as dichloroethane andchlorobenzene give reasonably satisfactory yields, but yields aregenerally better in a methylene chloride reaction medium. Solvents suchas acetonitrile and ethyl acetate generally give poor yields, whilereaction in solvents such as methanol or water/tetrahydrofuran givelittle of the desired product.

[0277] Further in accordance with the present invention, it has beendiscovered that numerous improvements in the synthesis of epoxymexrenonecan be realized by use of a trihaloacetamide rather than atrihaloacetonitrile as a peroxide activator for the epoxidationreaction. In accordance with a particularly preferred process, theepoxidation is carried out by reaction of the substrate of Formula IIAwith hydrogen peroxide in the presence of trichloroacetamide and anappropriate buffer. Preferably, the reaction is conducted in a pH in therange of about 3 to about 7, most preferably between about 5 and about7. However, despite these considerations, successful reaction has beenrealized outside the preferred pH ranges.

[0278] Especially favorable results are obtained with a buffercomprising dipotassium hydrogen phosphate, and/or with a buffercomprising a combination of dipotassium hydrogenphosphate and potassiumdihydrogen phosphate in relative proportions of between about 1:4 andabout 2:1, most preferably in the range of about 2:3. Borate buffers canalso be used, but generally give slower conversions than dipotassiumphosphate or K₂HPO₄ or K₂HPO₄/KH₂PO₄ mixtures. Whatever the makeup ofthe buffer, it should provide a pH in the range indicated above. Asidefrom the overall composition of the buffer or the precise pH it mayimpart, it has been observed that the reaction proceeds much moreeffectively if at least a portion of the buffer is comprised of dibasichydrogenphosphate ion. It is believed that this ion may participateessentially as a homogeneous catalyst in the formation of an adduct orcomplex comprising the promoter and hydroperoxide ion, the generation ofwhich may in turn be essential to the overall epoxidation reactionmechanism. Thus, the quantitative requirement for dibasichydrogenphosphate (preferably from K₂HPO₄) may be only a small catalyticconcentration. Generally, it is preferred that HPO₄ be present in aproportion of at least about 0.1 equivalents, e.g., between about 0.1and about 0.3 equivalents, per equivalent substrate.

[0279] The reaction is carried out in a suitable solvent, preferablymethylene chloride, but alternatively other halogenated solvents such aschlorobenzene or dichloroethane can be used. Toluene and mixtures oftoluene and acetonitrile have also been found satisfactory. Withoutcommitting to a particular theory, it is posited that the reactionproceeds most effectively in a two phase system in which a hydroperoxideintermediate is formed and distributes to the organic phase of low watercontent, and reacts with the substrate in the organic phase. Thus thepreferred solvents are those in which water solubility is low. Effectiverecovery from toluene is promoted by inclusion of another solvent suchas acetonitrile.

[0280] In the conversion of substrates of Formula II to products ofFormula I, toluene provides a process advantage since the substrates arefreely soluble in toluene and the products are not. Thus, the productprecipitates during the reaction when conversions reach the 40-50%range, producing a three phase mixture from which the product can beconveniently separated by filtration. Methanol, ethyl acetate,acetonitrile alone, THF and THF/water have not proved as to be aseffective as the halogenated solvents or toluene in carrying out theconversion of this step of the process.

[0281] While trichloroacetamide is a highly preferred reagent, othertrihaloacetamides such as trifluoroacetamide can also be used.Trihalomethylbenzamide, and other compounds having an arylene moietybetween the electron withdrawing trihalomethyl group and the carbonyl ofthe amide, may also be useful. 3,3,3-Trihalopropionamides may also beused, but with less favorable results. Generically, the peroxideactivator may correspond to the formula:

R^(o)C(O)NH₂

[0282] where R^(o) is a group having an electron withdrawing strength(as measured by sigma constant) at least as high as that of themonochloromethyl group. More particularly, the peroxide activator maycorrespond to the formula:

[0283] where X¹, X², and X³ are independently selected from among halo,hydrogen, alkyl, haloalkyl and cyano and cyanoalkyl, and R^(p) isselected from among arylene and —(CX⁴X⁵)_(n)—, where n is 0 or 1, atleast one of X¹, X², X³, X⁴ and X⁵ being halo or perhaloalkyl. Where anyof X¹, X², X³, X⁴ or X⁵ is not halo, it is preferably haloalkyl, mostpreferably perhaloalkyl. Particularly preferred activators include thosein which n is 0 and at least two of X¹, X² and X³ are halo; or in whichall of X¹, X², X³, X⁴ and X⁵ are halo or perhaloalkyl. Each of X¹, X²X³, X⁴ and X⁵ is preferably Cl or F, most preferably Cl, though mixedhalides may also be suitable, as may perchloralkyl or perbromoalkyl andcombinations thereof.

[0284] Preferably, the peroxide activator is present in a proportion ofat least about 1 equivalents, more preferably between about 1.5 andabout 2 equivalents, per equivalent of substrate initially present.Hydrogen peroxide should be charged to the reaction in at least modestexcess, or added progressively as the epoxidation reaction proceeds.Although the reaction consumes only one to two equivalents of hydrogenperoxide per mole of substrate, hydrogen peroxide is preferably chargedin substantial excess relative to substrate and activator initiallypresent. Without limiting the invention to a particular theory, it isbelieved that the reaction mechanism involves formation of an adduct ofthe activator and OOH—, that the formation of this reaction isreversible with the equilibrium favoring the reverse reaction, and thata substantial initial excess of hydrogen peroxide is therefore necessaryin order to drive the reaction in the forward direction. Temperature ofthe reaction is not narrowly critical, and may be effectively carriedout within the range of 0° to 100° C. The optimum temperature depends onthe selection of solvent. Generally, the preferred temperature isbetween about 20° C. and 30° C., but in certain solvents, e.g., toluenethe reaction may be advantageously conducted in the range of 60°-70° C.At 25° C., reaction typically requires less than 10 hours, typically 3to 6 hours. If needed additional activator and hydrogen peroxide may beadded at the end of the reaction cycle to achieve complete conversion ofthe substrate.

[0285] At the end of the reaction cycle, the aqueous phase is removed,the organic reaction solution is preferably washed for removal of watersoluble impurities, after which the product may be recovered by removalof the solvent. Before removal of solvent, the reaction solution shouldbe washed, at with least a mild to moderately alkaline wash, e.g.,sodium carbonate. Preferably, the reaction mixture is washedsuccessively with: a mild reducing solution such as a weak (e.g. 3% byweight) solution of sodium sulfite in water; an alkaline solution, e.g.,NaOH or KOH (preferably about 0.5N); an acid solution such as HCl(preferably about 1N); and a final neutral wash comprising water orbrine, preferably saturated brine to minimize product losses. Prior toremoval of the reaction solvent, another solvent such as an organicsolvent, preferably ethanol may be advantageously added, so that theproduct may be recovered by crystallization after distillation forremoval of the more volatile reaction solvent.

[0286] It should be understood that the novel epoxidation methodutilizing trichloroacetamide or other novel peroxide activator hasapplication well beyond the various schemes for the preparation ofepoxymexrenone, and in fact may be used for the formation of epoxidesacross olefinic double bonds in a wide variety of substrates subject toreaction in the liquid phase. The reaction is particularly effective inunsaturated compounds in which the olefinic carbons are tetrasubstitutedand trisubstituted, i.e., R^(a)R^(b)C═CR^(c)R^(d) andR^(a)R^(b)C═CR^(c)RH where R^(a) to R^(d) represent substituents otherthan hydrogen. The reaction proceeds most rapidly and completely wherethe substrate is a cyclic compound with a trisubstituted double, oreither a cyclic or acyclic compound with tetrasubstituted double bonds.Exemplary substrates for this reaction include Δ-9,11-canrenone, and

[0287] Because the reaction proceeds more rapidly and completely withtrisubstituted and tetrasubstituted double bonds, it is especiallyeffective for selective epoxidation across such double bonds incompounds that may include other double bonds where the olefinic carbonsare monosubstituted, or even disubstituted.

[0288] It should be further understood that the reaction may be used toadvantage in the epoxidation of monosubstituted or even disubstituteddouble bonds, such as the 11,12-olefin in various steroid substrates.However, because it preferentially epoxidizes the more highlysubstituted double bonds, e.g., the 9,11-olefin, with high selectivity,the process of this invention is especially effective for achieving highyields and productivity in the epoxidation steps of the various reactionschemes described elsewhere herein.

[0289] The improved process has been shown to be particularlyadvantageous application to the preparation of:

[0290] Multiple advantages have been demonstrated for the process of theinvention in which trichloroacetamide is used in place oftrichloroacetonitrile as the oxygen transfer reagent for the epoxidationreaction. The trichloroacetamide reagent system provides tightregiocontrol for epoxidation across trisubstituted double withdisubstituted and α,β-keto olefins in the same molecular structure.Thus, reaction yield, product profile and final purity are substantiallyenhanced. It has further been discovered that the substantial excessoxygen generation observed with the use of trihaloacetonitrile is notexperienced with trichloroacetamide, imparting improved safety to theepoxidation process. Further in contrast to the trichloroacetonitrilepromoted reaction, the trichloroacetamide reaction exhibits minimumexothermic effects, thus facilitating control of the thermal profile ofthe reaction. Agitation effects are observed to be minimal and reactorperformance more consistent, a further advantage over thetrichloroacetonitrile process. The reaction is more amenable to scaleupthan the trichloroacetonitrile promoted reaction. Product isolation andpurification is simple, there is no observable Bayer-Villager oxidationof carbonyl function (peroxide promoted conversion of ketone to ester)as experienced, e.g., using m-chloroperoxybenzoic acid or other peracidsand the reagent is inexpensive, readily available, and easily handled.

[0291] The novel epoxidation method of the invention is highly useful asthe concluding step of the synthesis of Scheme 1. In a particularlypreferred embodiment, the overall process of Scheme 1 proceeds asfollows:

[0292] The second of novel reaction schemes (Scheme 2) of this inventionstarts with canrenone or other substrate corresponding to Formula XIII

[0293] where —A—A—, R³, —B—B—, R⁸ and R⁹ are as defined in Formula VIII.In the first step of this process, the substrate of Formula XIII isconverted to a product of Formula XII

[0294] using a cyanidation reaction scheme substantially the same asthat described above for conversion of the substrate of Formula VIII tothe intermediate of Formula VII. Preferably, the substrate of FormulaXIII corresponds to Formula XIIIA

[0295] and the enamine product corresponds to Formula XIIA

[0296] in each of which —A—A—, —B—B—, Y¹, Y², and X are as defined inFormula XIII.

[0297] In the second step of scheme 2, the enamine of Formula XII ishydrolyzed to an intermediate diketone product of Formula XI

[0298] where —A—A—, R³, —B—B—, R⁸ and R⁹ are as defined in Formula VIII,using a reaction scheme substantially the same as that described abovefor conversion of the substrate of Formula VIII to the intermediate ofFormula VII. Preferably, the substrate of Formula XII corresponds toFormula XIIA

[0299] and the diketone product corresponds to Formula XIA

[0300] in each of which —A—A—, —B—B—, Y¹, Y², and X are as defined inFormula VIIIA.

[0301] Further in accordance with reaction scheme 2, the diketone ofFormula XI is reacted with an alkali metal alkoxide to form mexrenone orother product corresponding to Formula X,

[0302] in each of which —A—A—, R³, —B—B—, R⁸ and R⁹ are as defined inFormula VIII. R¹ is as defined in Formula V. The process is carried outusing substantially the same reaction scheme that is described above forthe conversion of the compounds of Formula VI to those of Formula V.Preferably, the substrate of Formula XI corresponds to Formula XIA

[0303] and the intermediate product corresponds to Formula XA

[0304] in each of which —A—A—, —B—B—, Y¹, Y², and X are as defined inFormula XIII. R¹ is as defined in Formula V.

[0305] Canrenone and other compounds of Formula X are next9α-hydroxylated by a novel bioconversion process to yield products ofFormula IX

[0306] where —A—A—, R³, —B—B—, R⁸ and R⁹ are as defined in Formula VIII,and R¹ is as defined in Formula V. Among the organisms that can be usedin this hydroxylation step are Nocardia conicruria ATCC 31548, Nocardiaaurentia ATCC 12674, Corynespora cassiicola ATCC 16718, Streptomyceshydroscopicus ATCC 27438, Mortierella isabellina ATCC 42613, Beauyriabassiana ATCC 7519, Penicillum purpurogenum ATCC 46581, Hypomyceschrysospermus IMI 109891, Thamnostylum piriforme ATCC 8992,Cunnignhamella blakesleeana ATCC 8688a, Cunninqnhamella echinulata ATCC3655, Cunninghamella elegans ATCC 9245, Trichothecium roseum ATCC 12543,Epicoccum humicola ATCC 12722, Saccharopolyspora eythrae ATCC 11635,Beauvria bassiana ATCC 13144, Arthrobacter simplex, Bacteriumcyclooxydans ATCC 12673, Cylindrocarpon radicicola ATCC 11011, Nocardiaaurentia ATCC 12674, Nocardia canicruria, Norcardia restrictus ATCC14887, Pseudomonas testosteroni ATCC 11996, Rhodococcus equi ATCC 21329,Mycobacterium fortuitum ATCC-6842, and Rhodococcus rhodochrous ATCC19150. The reaction is carried out substantially in the manner describedabove in connection with FIGS. 1 and 2. The process of FIG. 1 isparticularly preferred.

[0307] Growth media useful in the bioconversions preferably containbetween about 0.05% and about 5% by weight available nitrogen; betweenabout 0.5% and about 5% by weight glucose; between about 0.25% and about2.5% by weight of a yeast derivative; and between about 0.05%, and about0.5% by weight available phosphorus. Particularly preferred growth mediainclude the following:

[0308] soybean meal: between about 0.5% and about 3% by weight glucose;between about 0.1% and about 1% by weight soybean meal; between about0.05% and about 0.5% by weight alkali metal halide; between about 0.05%and about 560.5% by weight of a yeast derivative such as autolyzed yeastor yeast extract; between about 0.05% and about 0.5% by weight of aphosphate salt such as K₂HPO₄; pH=7;

[0309] peptone-yeast extract-glucose: between about 0.2% and about 2% byweight peptone; between about 0.05% and about 0.5% by weight yeastextract; and between about 2% and about 5% by weight glucose;

[0310] Mueller-Hinton: between about 10% and about 40% by weight beefinfusion; between about 0.35% and about 8.75% by weight casamino acids;between about 0.15% and about 0.7% by weight starch.

[0311] Fungi can be grown in soybean meal or peptone nutrients, whileactinomycetes and eubacteria can be grown in soybean meal (plus 0.5% to1% by weight carboxylic acid salt such as Na formate forbiotransformations) or in Mueller-Hinton broth.

[0312] The production of 11β-hydroxymexrenone from mexrenone byfermentation is discussed in Example 19.

[0313] The products of Formula IX are novel compounds, which may beseparated by filtration; washed with a suitable organic solvent, e.g.,ethyl acetate, and recrystallized from the same or a similar solvent.They have substantial value as intermediates for the preparation ofcompounds of Formula I, and especially of Formula IA. Preferably, thecompounds of Formula IX correspond to Formula IXA in which —A—A— and—B—B— are —CH₂—CH₂—, R³ is hydrogen, lower alkyl or lower alkoxy, and R⁸and R⁹ together constitute the 20-spiroxane ring:

[0314] In the next step of synthesis scheme 2, the product of Formula IXis reacted with a dehydration reagent to produce a compound of FormulaII

[0315] wherein —A—A—, R³, —B—B—, R⁸ and R⁹ are as defined in FormulaVIII, and R¹ is as defined in Formula V. Where the substrate correspondsto Formula IXA, the product is of Formula IIA

[0316] in each of which —A—A—, —B—B—, Y¹, Y², and X are as defined inFormula XIII and R¹ is as defined in Formula V.

[0317] In the final step of this synthesis scheme, the product ofFormula II is converted to that of Formula I by epoxidation inaccordance with the method described in U.S. Pat. No. 4,559,332; orpreferably by the novel epoxidation method of the invention as describedhereinabove.

[0318] In a particularly preferred embodiment, the overall process ofScheme 2 proceeds as follows:

[0319] The synthesis in this case begins with a substrate correspondingto Formula XX

[0320] where —A—A— and R³ are as defined in Formula VIII, —B—B— is asdefined in Formula VIII except that neither R⁶ nor R⁷ is part of a ringfused to the D ring at the 16,17 positions, and R²⁶ is lower alhyl,preferably methyl. Reaction of the substrate of Formula XX with asulfonium ylide produces the epoxide intermediate corresponding toFormula XIX

[0321] wherein —A—A—, R³, —B—B—, and R²⁶ are as defined in Formula XX.

[0322] In the next step of synthesis scheme 3, the intermediate ofFormula XIX is converted to a further intermediate of Formula XVIII

[0323] wherein —A—A—, R³, and —B—B— are as defined in Formula XX. Inthis step, Formula XIX substrate is converted to Formula XVIIIintermediate by reaction with NaCH(COOEt)₂ in the presence of a base ina solvent. Exposure of the compound of Formula XVIII to heat water andan alkali halide produces a decarboxylated intermediate compoundcorresponding to Formula XVII

[0324] wherein —A—A—, R³, and —B—B— are as defined in Formula XX. Theprocess for conversion of the compound of Formula XX to the compound ofFormula XVII corresponds essentially to that described in U.S. Pat. Nos.3,897,417, 3,413,288 and 3,300,489, which are expressly incorporatedherein by reference. While the substrates differ, the reagents,mechanisms and conditions for introduction of the 17-spirolactone moietyare essentially the same.

[0325] Reaction of the intermediate of Formula XVII with adehydrogenation reagent yields the further intermediate of Formula XVI.

[0326] where —A—A—, R³ and —B—B— are as defined above.

[0327] Typically useful dehydrogenation reagents includedichlorodicyanobenzoquinone (DDQ) and chloranil(2,3,5,6-tetrachloro-p-benzoquinone). Alternatively, the dehydrogenationcould be achieved by a sequential halogenation at the carbon-6 followedby dehydrohalogenation reaction.

[0328] The intermediate of Formula XVI is next converted to the enamineof Formula XV

[0329] wherein —A—A—, R³, and —B—B— are as defined in Formula XX.Conversion is by cyanidation essentially in the manner described abovefor the conversion of the 11α-hydroxy compound of Formula VIII to theenamine of Formula VII. Typically, the cyanide ion source may be analkali metal cyanide. The base is preferably pyrrolidine and/ortetramethylguanidine. A methanol solvent may be used.

[0330] The products of Formula XV are novel compounds, which may beisolated by chromatography. These and other novel compounds of FormulaAXV have substantial value as intermediates for the preparation ofcompounds of Formula I, and especially of Formula IA. Compounds ofFormula AXV correspond to the structure

[0331] where —A—A—, —B—B—, R³, R⁸ and R⁹ are as defined above. In themost preferred compounds of Formula XV, and —A—A— and —B—B— are—CH₂—CH₂—.

[0332] In accordance with the hydrolysis described above for producingthe diketone compounds of Formula VI, the enamines of Formula XV may beconverted to the diketones of Formula XIV

[0333] wherein —A—A—, R³, and —B—B— are as defined in Formula XX.Particularly preferred for the synthesis of epoxymexrenone are thosecompounds of Formula XIV which also fall within the scope of FormulaVIA.

[0334] The products of Formula XIV are novel compounds, which may beisolated by precipitation. These and other novel compounds of FormulaAXIV have substantial value as intermediates for the preparation ofcompounds of Formula I, and especially of Formula IA. Compounds ofFormula AXIV correspond to the structure

[0335] where —A—A—, —B—B—, R³, R⁸ and R⁹ are as defined above. In themost preferred compounds of Formula AXIV and XIV, —A—A— and —B—B— are—CH₂—CH₂—.

[0336] The compounds of Formula XIV are further converted to compoundsof Formula XXXI using essentially the process described above forconverting the diketone of Formula VI to the hydroxyester of Formula V.In this instance, it is necessary to isolate the intermediate XXXI

[0337] before further conversion to a product of Formula XXXII

[0338] wherein —A—A— and —B—B— are as defined in Formula XX. Preferredcompounds of Formula XXXI are those which fall within Formula IIA. Thecompounds of Formula XXXI are converted to compounds of Formula XXXIIusing the method described hereinabove or in U.S. Pat. No. 4,559,332. Ina particularly preferred embodiment, the overall process of Scheme 3proceeds as follows:

[0339] The first three steps of Scheme 4 are the same as those of Scheme3, i.e., preparation of an intermediate of Formula XVII starting with acompound corresponding to Formula XX.

[0340] Thereafter, the intermediate of Formula XVII is epoxidized, forexample, using the process of U.S. Pat. No. 4,559,332 to produce thecompound of Formula XXIV

[0341] wherein —A—A—, R³, and —B—B— are as defined in Formula XX.However, in a particularly preferred embodiment of the invention, thesubstrate of Formula XVII is epoxidized across the 9,11-double bondusing an oxidation reagent comprising an amide type peroxide activator,most preferably trichloroacetamide, according to the process asdescribed above in Scheme 1 for the conversion of the enester of FormulaII to the product of Formula I. The conditions and proportions ofreagents for this reaction are substantially as described for theconversion of the Formula II enester to epoxymexrenone.

[0342] It has been found that the epoxidation of the substrate ofFormula XVII can also be effected in very good yield using a peracidsuch as, for example, m-chloroperoxybenzoic acid. However, thetrichloroacetamide reagent provides superior results in minimizing theformation of Bayer-Villager oxidation by-product. The latter by-productcan be removed, but this requires trituration from a solvent such asethyl acetate, followed by crystallization from another solvent such asmethylene chloride. The epoxy compound of Formula XXIV is dehydrogenatedto produce a double bond between the 6- and 7-carbons by reaction with adehydrogenation (oxidizing) agent such as DDQ or chloranil, or using thebromination/dehydrobromination (or otherhalogenation/dehydrohalogenation) sequence, to produce another novelintermediate of Formula XXIII

[0343] wherein —A—A—, and —B—B— are as defined in Formula XX.Particularly preferred compounds of Formula XXIII are those in which—A—A— and —B—B— are as defined in Formula XIII.

[0344] While direct oxidation is effective for the formation of theproduct of Formula XXIII, the yields are generally low. Preferably,therefore, the oxidation is carried out in two steps, first halogenatingthe substrate of Formula XXIV at the C-6 position, thendehydrohalogenating to the 6,7-olefin. Halogenation is preferablyeffected with an N-halo organic reagent such as, for example,N-bromosuccinamide. Bromination is carried out in a suitable solventsuch as, for example, acetonitrile, in the presence of halogenationpromoter such as benzoyl peroxide. The reaction proceeds effectively ata temperature in the range of about 50° to about 100° C., convenientlyat atmospheric reflux temperature in a solvent such as carbontetrachloride, acetonitrile or mixture thereof. However, reaction from 4to 10 hours is typically required for completion of the reaction. Thereaction solvent is stripped off, and the residue taken up awater-immiscible solvent, e.g., ethyl acetate. The resulting solution iswashed sequentially with a mild alkaline solution (such as an alkalimetal bicarbonate) and water, or preferably saturated brine to minimizeproduct losses, after which the solvent is stripped and a the residuetaken up in another solvent (such as dimethylformamide) that is suitablefor the dehydrohalogenation reaction.

[0345] A suitable dehydrohalogenation reagent, e.g.,1,4-diazabicyclo[2,2,2]octane (DABCO) is added to the solution, alongwith an alkali metal halide such as LiBr, the solution heated to asuitable reaction temperature, e.g., 60° to 80° C., and reactioncontinued for several hours, typically 4 to 15 hours, to complete thedehydrobromination. Additional dehydrobromination reagent may be addedas necessary during the reaction cycle, to drive the reaction tocompletion. The product of Formula XXIII may then be recovered, e.g., byadding water to precipitate the product which is then separated byfiltration and preferably washed with additional amounts of water. Theproduct is preferably recrystallized, for example fromdimethylformamide.

[0346] The products of Formula XXIII, such as 9,11-epoxycanrenone, arenovel compounds, which may be isolated by extraction/crystallization.They have substantial value as intermediates for the preparation ofcompounds of Formula I, and especially of Formula IA. For example, theymay be used as substrates for the preparation of compounds of FormulaXXII. In the most preferred compounds of Formula XXIII, and —A—A— and—B—B— are —CH₂—CH₂—.

[0347] Using substantially the process described above for thepreparation of compounds of Formula VII, the compounds of Formula XXIIIare reacted with cyanide ion to produce novel epoxyenamine compoundscorresponding to Formula XXII

[0348] wherein —A—A—, R³, and —B—B— are as defined in Formula XX.Particularly preferred compounds of Formula XXII are those in which—A—A— and —B—B— are as defined in Formula XIII.

[0349] The products of Formula XXII are novel compounds, which may beisolated by precipitation and filtration. They have substantial value asintermediates for the preparation of compounds of Formula I, andespecially of Formula IA. In the most preferred compounds of FormulaXXII, and —A—A— and —B—B— are —CH₂—CH₂—

[0350] Using substantially the process described above for preparationof compounds of Formula VI, the epoxyenamine compounds of Formula XXIIare converted to novel epoxydiketone compounds of Formula XXI.

[0351] The products of Formula XXI are novel compounds, which may beisolated by precipitation and filtration. They have substantial value asintermediates for the preparation of compounds of Formula I, andespecially of Formula IA. Particularly preferred compounds of FormulaXXI are those in which —A—A— and —B—B— are as defined in Formula XIII.In the most preferred compounds of Formula XXI, and —A—A— and —B—B— are—CH₂—CH₂—.

[0352] Compounds of Formula XXI are converted to compounds of FormulaXXXII using the epoxidation process described hereinabove or the processof U.S. Pat. No. 4,559,332. In a particularly preferred embodiment, theoverall process of Scheme 4 proceeds as follows:

[0353] The process of scheme 5 begins with a substrate corresponding toFormula XXIX

[0354] wherein —A—A—, and —B—B— are as defined in Formula XX. Thissubstrate is converted to a product of Formula XXVIII

[0355] by reaction with trimethylorthoformate. wherein —A—A—, R³, and—B—B— are as defined in Formula XX. Following the formation of FormulaXXVIII, the compounds of Formula XXIX are converted to compounds ofFormula XXVII using the method described above for conversion of thesubstrate of Formula XX to Formula XVII. Compounds of Formula XXVII havethe structure:

[0356] wherein —A—A—, and —B—B— are as defined in Formula XX, and R^(x)is any of the common hydroxyl protecting groups.

[0357] Using the method described above for the preparation of compoundsof Formula XVI, compounds of Formula XXVII are oxidized to yield novelcompounds corresponding to Formula XXVI

[0358] wherein —A—A—, and —B—B— are as defined in Formula XX.Particularly preferred compounds of Formulae XXIX, XXVIII, XXVII andXXVI are those in which —A—A— and —B—B— are as defined in Formula XIII.

[0359] The products of Formula XXVI are novel compounds, which may beisolated by precipitation/filtration. They have substantial value asintermediates for the preparation of compounds of Formula I, andespecially of Formula IA. Particularly preferred compounds of FormulaXXVI are those in which —A—A— and —B—B— are as defined in Formula XIII.In the most preferred compounds of Formula XXVI, and —A—A— and —B—B— are—CH₂—CH₂—.

[0360] Using the method defined above for cyanidation of compounds ofFormula VIII, the novel intermediates of Formula XXVI are converted tothe novel 9-hydroxyenamine intermediates of Formula XXV

[0361] wherein —A—A—, R³, and —B—B— are as defined in Formula XX.

[0362] The products of Formula XXV are novel compounds, which may beisolated by precipitation/filtration. They have substantial value asintermediates for the preparation of compounds of Formula I, andespecially of Formula IA. Particularly preferred compounds of FormulaXXV are those in which —A—A— and —B—B— are as defined in Formula XIII.In the most preferred compounds of Formula XXVI, and —A—A— and —B—B— are—CH₂—CH₂—.

[0363] Using essentially the conditions described above for thepreparation of the diketone compounds of Formula VI, the9-hydroxyenamine intermediates of Formula XXV are converted to thediketone compounds of Formula XIV. Note that in this instance thereaction is effective for simultaneous hydrolysis of the enaminestructure and dehydration at the 9,11 positions to introduce the 9,11double bond. The compound of Formula XIV is then converted to thecompound of Formula XXXI, and thence to the compound of Formula XIII,using the same steps that are described above in scheme 3.

[0364] In a particularly preferred embodiment, the overall process ofScheme 5 proceeds as follows:

[0365] Scheme 6 provides an advantageous method for the preparation ofepoxymexrenone and other compounds corresponding to Formula I, startingwith 11α-hydroxylation of androstendione or other compound of FormulaXXXV

[0366] wherein —A—A—, R³, and —B—B— are as defined in Formula XIII,producing an intermediate corresponding to the Formula XXXVI

[0367] where —A—A—, R³, and —B—B— are as defined in Formula XIII. Exceptfor the selection of substrate, the process for conducting the11α-hydroxylation is essentially as described hereinabove for Scheme 1.The following microorganisms are capable of carrying out the11α-hydroxylation of androstendione or other compound of Formula XXXV:

[0368]Aspergillus ochraceus NRRL 405 (ATCC 18500);

[0369]Aspergillus niger ATCC 11394;

[0370]Aspergillus nidulans ATCC 11267;

[0371]Rhizopus oryzae ATCC 11145;

[0372]Rhizopus stolonifer ATCC 6227b;

[0373]Trichothecium roseum ATCC 12519 and ATCC 8685.

[0374] 11α-Hydroxyandrost-4-ene-3,17-dione, or other compound of FormulaXXXVI, is next converted to 11α-hydroxy-3,4-enol ether of Formula (101):

[0375] where —A—A—, R³, and —B—B—, are as defined in Formula XIII andR¹¹ is methyl or other lower alkyl (C₁ to C₄), by reaction with anetherifying reagent such as trialkyl orthoformate in the presence of anacid catalyst. To carry out this conversion, the 11α-hydroxy substrateis acidified by mixing with an acid such as, e.g., benzene sulfonic acidhydrate or toluene sulfonic acid hydrate and dissolved in a loweralcohol solvent, preferably ethanol. A trialkyl orthoformate, preferablytriethyl orthoformate is introduced gradually over a period of 5 to 40minutes while maintaining the mixture in the cold, preferably at about0° C. to about 15° C. The mixture is then warmed and the reactioncarried out at a temperature of between 20° C. and about 60° C.Preferably the reaction is carried out at 30° to 50° C. for 1 to 3hours, then heated to reflux for an additional period, typically 2 to 6hours, to complete the reaction. Reaction mixture is cooled, preferablyto 0° to 15°, preferably about 5° C., and the solvent removed undervacuum.

[0376] Using the same reaction scheme as described in Scheme 3, above,for the conversion of the compound of Formula XX to the compound ofFormula XVII, a 17-spirolactone moiety of Formula XXXIII is introducedinto the compound of Formula 101. For example, the Formula 101 substratemay be reacted with a sulfonium ylide in the presence of a base such asan alkali metal hydroxide in a suitable solvent such as DMSO, to producean intermediate corresponding to Formula 102:

[0377] where —A—A—, R³, R¹¹, and —B—B— are as defined in Formula 101.The intermediate of Formula 102 is then reacted with a malonic aciddiester in the presence of an alkali metal alkoxide to form the fivemembered spirolactone ring and produce the intermediate of Formula 103

[0378] where —A—A—, R³, R¹¹, R¹², and —B—B— are as defined in FormulaXIII. Finally, the compound of Formula 103 in a suitable solvent, suchas dimethylformamide, is subjected to heat in the presence of an alkalimetal halide, splitting off the alkoxycarbonyl moiety and producing theintermediate of Formula 104:

[0379] where again —A—A—, R³, R¹¹ and —B—B— are as defined in FormulaXIII.

[0380] Next the 3,4-enol ether compound 104 is converted to the compoundof Formula XXIII, i.e., the compound of Formula VIII in which R⁸ and R⁹together form the moiety of Formula XXXIII. This oxidation step iscarried out in essentially the same manner as the oxidation step forconversion of the compound of Formula XXIV to the intermediate ofFormula XXIII in the synthesis of Scheme 4. Direct oxidation can beeffected using a reagent such as2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) ortetrachlorobenzoquinone (chloranil), or preferably a two stage oxidationis effected by first brominating, e.g., with an N-halo brominating agentsuch as N-bromosuccinamide or 1,3-dibromo-5,5-dimethyl hydantoin (DBDMH)and then dehydrobrominating with a base, for example with DABCO in thepresence of LiBr and heat. Where NBS is used for bromination, an acidmust also be employed to convert 3-enol ether to the enone. DBDMH, anionic rather than free radical bromination reagent, is effective byitself for bromination and conversion of the enol ether to the enone.

[0381] The compound of Formula VIII is then converted to epoxymexrenoneor other compound of Formula I by the steps described hereinabove forScheme 1.

[0382] Each of the intermediates of Formulae 101, 102, 103, and 104 is anovel compound having substantial value as an intermediate forepoxymexrenone or other compounds of Formulae IA and I. In each of thecompounds of Formulae 101, 102, 103, and 104 —A—A— and —B—B— arepreferably —CH₂—CH₂— and R³ is hydrogen, lower alkyl or lower alkoxy.Most preferably, the compound of Formula 101 is3-ethoxy-11α-hydroxyandrost-3,5-dien-17-one, the compound of Formula 102is 3-ethoxyspiro[androst-3,5-diene-17β,2′-oxiran]-11α-ol, the compoundof Formula 103 is ethyl hydrogen3-ethoxy-11α-17α-dihydroxypregna-3,5-diene-21,21-dicarboxylate,gamma-lactone, and the compound of Formula 104 is3-ethoxy-11α-17α-dihydroxypregna-3,5-diene-21-carboxylic acid,gamma-lactone.

[0383] In a particularly preferred embodiment, the overall process ofScheme 6 proceeds as follows:

[0384] Scheme 7 provides for the synthesis of epoxymexrenone and othercompounds of Formula I using a starting substrate comprisingβ-sitosterol, cholesterol, stigmasterol or other compound of FormulaXXXVII

[0385] where —A—A—, R³, and —B—B— are as defined in Formula XIII, D—D is—CH₂—CH₂— or —CH═CH—, and each of R¹³, R¹⁴, R¹⁵ and R¹⁶ is independentlyselected from among hydrogen or C₁, to C₄ alkyl.

[0386] In the first step of the synthesis 11α-hydroxyandrostendione orother compound of Formula XXXV is prepared by bioconversion of thecompound of Formula XXXVII. The bioconversion process is carried outsubstantially in accordance with the method described hereinabove forthe 11α-hydroxylation of canrenone (or other substrate of Formula XIII).

[0387] In the synthesis 11α-hydroxyandrostendione,4-androstene-3,17-dione is initially prepared by bioconversion of thecompound of Formula XXXVII. This initial bioconversion may be carriedout in the manner described in U.S. Pat. No. 3,759,791, which isexpressly incorporated herein by reference. Thereafter,4-androstene-3,17-dione is converted to 11α-hydroxyandrostenedionesubstantially in accordance with the method described hereinabove forthe 11α-hydroxylation of canrenone (or other substrate of Formula XIII).

[0388] The remainder of the synthesis of Scheme 7 is identical to Scheme6. In a particularly preferred embodiment, the overall process of Scheme7 proceeds as follows:

[0389] The methods, processes and compositions of the invention, and theconditions and reagents used therein, are further described in thefollowing examples.

EXAMPLE 1

[0390] Slants were prepared with a growth medium as set forth in Table 1TABLE 1 Y P D A (medium for slants and plates) yeast extract 20 gpeptone 20 g glucose 20 g agar 20 g distilled water, q.s. to 1000 ml pHas is 6.7 adjust at pH 5 with H₃PO₄ 10% w/v Distribute for slants: 7.5ml in 180 × 18 mm tubes for plates (10 cm of φ) 25 ml in 200 × 20 mmtubes sterilize at 120° C. for 20 minutes pH after sterilization:5

[0391] To produce first generation cultures, a colony of Aspergillusochraceus was suspended in distilled water (2 ml) in a test tube; and0.15 ml aliquots of this suspension applied to each of the slants thathad been prepared as described above. The slants were incubated forseven days at 25° C., after which the appearance of the surface culturewas that of a white cottony mycelium. The reverse was pigmented inorange in the lower part, in yellow-orange in the upper part.

[0392] The first generation slant cultures were suspended in a sterilesolution (4 ml) containing Tween 80 nonionic surfactant (3% by weight),and 0.15 ml aliquots of this suspension were used to inoculate secondgeneration slants that had been prepared with the growth medium setforth in Table 2 TABLE 2 (for second generation and routine slants) maltextract 20 g peptone 1 g glucose 20 g agar 20 g distilled water q.s. to1000 ml pH as is 5.3 distribute in tubes (180 × 18 mm) ml 7.5 sterilizeat 120° C. for 20 minutes

[0393] The second generation slants were incubated for 10 days at 25°C., producing a heavy mass of golden-colored spores; reverse pigmentedin brown orange.

[0394] A protective medium was prepared having the composition set forthin Table 3. TABLE 3 PROTECTIVE MEDIUM Skim milk 10 g distilled water 100ml In a 250 ml flask containing 100 ml of distilled water at 50° C., addskim milk. Sterilize at 120° C. for 15 minutes. Cool at 33° C. and usebefore the day is over

[0395] Cultures from five of the second generation slants were suspendedin the protective solution (15 ml) in a 100 ml flask. The suspension wasdistributed in aliquots (0.5 ml each) among 100×10 mm tubes forlyophilization. These were pre-frozen at −70° to −80° C. in anacetone/dry ice bath for 20 minutes, then transferred immediately to adrying room pre-cooled to −40° to −50° C. The pre-frozen aliquots werelyophilized at a residual pressure of 50 μHg and ≦−30° C. At the end ofthe lyophilization, two to three granules of sterile silica gel wereadded to each tube with moisture indicator and flame seal.

[0396] To obtain mother culture slants suitable for industrial scalefermentation, a single aliquot of lyophilized culture, which had beenprepared in the manner described above, was suspended in distilled water(1 ml) and 0.15 ml aliquots of the suspension were used to inoculateslants that had been provided with a growth medium having thecomposition set forth in Table 2. The mother slants were incubated forseven days at 25° C. At the end of incubation, the culture developed onthe slants was preserved at 4° C.

[0397] To prepare a routine slant culture, the culture from a motherslant was suspended in a sterile solution (4 ml) containing Tween 80 (3%by weight) and the resulting suspension distributed in 0.15 ml aliquotsamong slants which had been coated with the growth medium described inTable 2. The routine slant cultures may be used to inoculate the primaryseed flasks for laboratory or industrial fermentations.

[0398] To prepare a primary seed flask culture, the culture from aroutine slant, which had been prepared as described above, was removedand suspended in a solution (10 ml) containing Tween 80 (3% by weight).A 0.1 aliquot of the resulting suspension was introduced into a 500 mlbaffled flask containing a growth medium having the composition setforth in Table 4. TABLE 4 (for primary and transformation flask cultureand round bottomed flask) glucose 20 g peptone 20 g yeast autolysate 20g distilled water q.s to pH as is 5.2 adjust at pH 5.8 with NaOH 20%distribute in 500 ml baffled flask 100 ml distribute in 2000 ml roundbottomed flasks (3 baffles) 500 ml sterilize 120° C. × 20 min. pH aftersterilization about 5.7

[0399] The seed flask was incubated on a rotating shaker (200 rpm, 5 cmdisplacement) for 24 hours at 28° C., thereby producing a culture in theform of pellet-like mycelia having diameters of 3 to 4 mm. Onmicroscopic observation, the seed culture was found to be a pureculture, with synnematic growth, with big hyphae and well twisted. ThepH of the suspension was 5.4 to 5.6. PMV was 5 to 8% as determined bycentrifugation (3000 rpm×5 min.).

[0400] A transformation flask culture was prepared by inoculating agrowth medium (100 ml) having the composition set forth Table 4 in asecond 500 ml shaker flask with biomass (1 ml) from the seed cultureflask. The resulting mixture was incubated on a rotating shaker (200rpm, 5 cm displacement) for 18 hours at 28° C. The culture was examinedand found to comprise pellet like mycelia with a 3-4 mm diameter. Onmicroscopic examination, the culture was determined to be a pureculture, with synnematic and filamentous growth in which the apicalcells were full of cytoplasm and the olden cells were little vacuolated.The pH of the culture suspension was 5 to 5.2 and the PMV was determinedby centrifugation to be between 10% and 15%. Accordingly, the culturewas deemed suitable for transformation of canrenone to11α-hydroxycanrenone.

[0401] Canrenone (1 g) was micronized to about 5μ and suspended insterile water (20 ml). To this suspension were added: a 40% (w/v)sterile glucose solution; a 16% (w/v) sterile solution of autolyzedyeast; and a sterile antibiotic solution; all in the proportionsindicated for 0 hours reaction time in Table 5. The antibiotic solutionhad been prepared by dissolving kanamicyn sulfate (40 mg), tetracyclineHCl (40 mg) and cefalexin (200 mg) in water (100 ml). The steroidsuspension, glucose solution, and autolyzed yeast solution were addedgradually to the culture contained in the shaker flask. TABLE 5Indicative Additions of Steroid and Solutions (additives andantibiotics) in the Course of Bioconversion of Canrenone in Shake Flaskyeast auto- anti- Reaction Steroid Suspension glucose lised biotic timeapprox. solution sol. solution hours ml mg. ml ml. ml 0 1 50 1 0.5 1 8 2100 2 1 24 2 100 1 0.5 1 32 5 250 2 1 48 2 100 1 0.5 1 56 5 250 2 1 72 3150 1 0.5 1 90

[0402] As reaction proceeded, the reaction mixture was periodicallyanalyzed to determine glucose content, and by thin layer chromatographyto determine conversion to 11α-hydroxycanrenone. Additional canrenonesubstrate and nutrients were added to the fermentation reaction mixtureduring the reaction at rates controlled to maintain the glucose contentin the range of about 0.1% by weight. The addition schedule for steroidsuspension, glucose solution, autolyzed yeast solution and antibioticsolution is set forth in Table 5. The transformation reaction continuedfor 96 hours at 25° C. on a rotary shaker (200 rpm and 5 cmdisplacement). The pH ranged between 4.5 and 6 during the fermentation.Whenever the PMV rose to or above 60%, a 10 ml portion of broth culturewas withdrawn and replaced with 10 ml distilled water. The disappearanceof canrenone and appearance of 11α-hydroxycanrenone were monitoredduring the reaction by sampling the broth at intervals of 4, 7, 23, 31,47, 55, 71, 80, and 96 hours after the start of the fermentation cycle,and analyzing the sample by TLC. The progress of the reaction asdetermined from these samples is set forth in Table 6 TABLE 6 TimeCourse of Bioconversion of Canrenone in Shake Flask Transformation RatioTime Canrenone Rf. 11αhydroxy Canrenone hours RF. = 0.81 RF. = 0.29 0100 0.0 4 50 50 7 20 80 23 20 80 31 30 70 47 20 80 55 30 70 71 25 75 8015 85 96 ˜10 ˜90

EXAMPLE 2

[0403] A primary seed flask culture was prepared in the manner describedin Example 1. A nutrient mixture was prepared having the composition setforth in Table 7 TABLE 7 For Transformation Culture in 10 l glassfermenter quantity g/l glucose 80 g 20 peptone 80 g 20 yeast autolised80 g 20 antifoam SAG 471 0.5 g  deionized water q.s. to  4 l sterilizethe empty fermenter for 30 minutes at 130° C. load it with 3 l ofdeionized water, heat at 40° C. add while stirring the components of themedium stir for 15 minutes, bring to volume of 3.9 l pH as is 5.1 adjustof 5.8 with NaOH 20% w/v sterilize at 120° C. × 20 minutes pH aftersterilization 5.5-5.7

[0404] An initial charge of this nutrient mixture (4 L) was introducedinto a transformation fermenter of 10 L geometric volume. The fermenterwas of cylindrical configuration with a height to diameter ratio of2.58. It was provided with a 400 rpm turbine agitator having two No. 2disk wheels with 6 blades each. The external diameter of the impellerswas 80 mm, each of the blades was 25 mm in radial dimension and 30 mmhigh, the upper wheel was positioned 280 mm below the top of the vessel,the lower wheel was 365 mm below the top, and baffles for the vesselwere 210 mm high and extended radially inwardly 25 mm from the interiorvertical wall of the vessel.

[0405] Seed culture (40 ml) was mixed with the nutrient charge in thefermenter, and a transformation culture established by incubation for 22hours at 28° C., and an aeration rate of 0.5 l/l-min. at a pressure of0.5 kg/cm². At 22 hours, the PMV of the culture was 20-25% and the pH 5to 5.2.

[0406] A suspension was prepared comprising canrenone (80 g) in sterilewater (400 ml), and a 10 ml portion added to the mixture in thetransformation fermenter. At the same time a 40% (w/v) sterile glucosesolution, a 16% (w/v) sterile solution of autolyzed yeast, and a sterileantibiotic solution were added in the proportions indicated in Table 8at 0 hours reaction time. The antibiotic solution was prepared in themanner described in Example 1. TABLE 8 Indicative Additions of Steroidand Solutions (additives and antibiotics) in the Course of Bioconversionof Canrenone in 10 l Glass Fermenter Steroid yeast anti- ReactionSuspension glucose autolised biotic time approx solution solutionsolution hours ml gr ml ml ml 0 10 4 25 12.5 40 4 25 12.5 8 10 4 25 12.512 25 12.5 16 10 4 25 12.5 20 25 12.5 24 10 4 25 12.5 40 28 10 4 25 12.532 12.5 5 25 12.5 36 12.5 5 25 12.5 40 12.5 5 25 12.5 44 12.5 5 25 12.548 12.5 5 25 12.5 40 52 12.5 5 25 12.5 56 12.5 5 25 12.5 60 12.5 5 2512.5 64 12.5 5 25 12.5 68 12.5 5 25 12.5 72 12.5 5 25 12.5 40 76 12.5 525 12.5 80 84 88

[0407] As reaction proceeded, the reaction mixture was periodicallyanalyzed to determine glucose content, and by thin layer chromatographyto determine conversion to 11α-hydroxycanrenone. Based on TLC analysisof reaction broth samples as described hereinbelow, additional canrenonewas added to the reaction mixture as canrenone substrate was consumed.Glucose levels were also monitored and, whenever glucose concentrationdropped to about 0.05% by weight or below, supplemental glucose solutionwas added to bring the concentration up to about 0.25% by weight.Nutrients and antibiotics were also added at discrete times during thereaction cycle. The addition schedule for steroid suspension, glucosesolution, autolyzed yeast solution and antibiotic solution is set forthin Table 8. The transformation reaction continued for 90 hours at anaeration rate of 0.5 vol. air per vol. liquid per minute (vvm) at apositive head pressure of 0.3 kg/cm². The temperature was maintained at28° C. until PVM reached 45%, then decreased to 26° C. and maintained atthat temperature as PVM grew from 45% to 60%, and thereafter controlledat 24° C. The initial agitation rate was 400 rpm, increasing to 700 rpmafter 40 hours. The pH was maintained at between 4.7 and 5.3 byadditions of 2M orthophosphoric acid or 2M NaOH, as indicated. Foamingwas controlled by adding a few drops of Antifoam SAG 471 as foamdeveloped. The disappearance of canrenone and appearance of11α-hydroxycanrenone were monitored at 4 hour intervals during thereaction by TLC analysis of broth samples. When most of the canrenonehad disappeared from the broth, additional increments were added.

[0408] After all canrenone additions had been made, the reaction wasterminated when TLC analysis showed that the concentration of canrenonesubstrate relative to 11α-hydroxycanrenone product had dropped to about5%.

[0409] At the conclusion of the reaction cycle, the fermentation brothwas filtered through cheese cloth for separation of the mycelium fromthe liquid broth. The mycelia fraction was resuspended in ethyl acetateusing about 65 volumes (5.2 liters) per gram canrenone charged over thecourse of the reaction. The suspension of mycelia in ethyl acetate wasrefluxed for one hour under agitation, cooled to about 20° C., andfiltered on a Buchner. The mycelia cake was washed sequentially withethyl acetate (5 vol. per g canrenone charge; 0.4 L) and deionized water(500 ml) to displace the ethyl acetate extract from the cake. The filtercake was discarded. The rich extract, solvent washing and water washingwere collected in a separator, then allowed to stand for 2 hours forphase separation.

[0410] The aqueous phase was then discarded and the organic phaseconcentrated under vacuum to a residual volume of 350 ml. The stillbottoms were cooled to 15° C. and kept under agitation for about onehour. The resulting suspension was filtered to remove the crystallineproduct, and the filter cake was washed with ethyl acetate (40 ml).After drying, the yield of 11α-hydroxycanrenone was determined to be 60g.

EXAMPLE 3

[0411] A spore suspension was prepared from a routine slant in themanner described in Example 1. In a 2000 ml baffled round bottomed flask(3 baffles, each 50 mm×30 mm), an aliquot (0.5 ml) of the sporesuspension was introduced into a nutrient solution (500 ml) having thecomposition set forth in Table 4. The resulting mixture was incubated inthe flask for 24 hours at 25° C. on an alternating shaker (120 strokesper min.; displacement 5 cm), thereby producing a culture which, onmicroscopic examination, was observed to appear as a pure culture withhyphae well twisted. The pH of the culture was between about 5.3 and5.5, and the PMV (as determined by centrifugation at 3000 rpm for 5min.) was 8 to 10%.

[0412] Using the culture thus prepared, a seed culture was prepared in astainless steel fermenter of vertical cylindrical configuration, havinga geometric volume of 160 L and an aspect ratio of 2.31 (height=985 mm;diameter=425 mm). The fermenter was provided with a disk turbine typeagitator having two wheels, each wheel having six blades with anexternal diameter of 240 mm, each blade having a radial dimension of 80mm and a height of 50 mm. The upper wheel was positioned at a depth of780 mm from the top of the fermenter, and the second at a depth of 995mm. Vertical baffles having a height of 890 mm extended radiallyinwardly 40 mm from the interior vertical wall of the fermenter. Theagitator was operated at 170 rpm. A nutrient mixture (100 L) having thecomposition set forth in Table 9 was introduced into the fermenter,followed by a portion of preinoculum (1 L) prepared as described aboveand having a pH of 5.7. TABLE 9 For Vegetative Culture in 160 LFermenter About 8 L are needed to Seed Productive fermenter Quantity g/Lglucose 2 kg 20 peptone 2 kg 20 yeast autolysed 2 kg 20 antifoam SAG 4710.010 Kg traces deionized water q.s. to 100 L sterilize the emptyfermenter for 1 hour at 130° C. load it with 6 L of deionized water;heat at 40° C. add while stirring the components of the medium stir for15 minutes, bring to volume of 95 L sterilization at 121° C. for 30minutes post sterilization pH is 5.7 add sterile deionized water to 100L

[0413] The inoculated mixture was incubated for 22 hours at an aerationrate of 0.5 L/L-min. at a head pressure of 0.5 kg/cm². The temperaturewas controlled at 28° C. until PMV reached 25%, and then lowered to 25°C. The pH was controlled in the range of 5.1 to 5.3. Growth of myceliumvolume is shown in Table 10, along with pH and dissolved oxygen profilesof the seed culture reaction. TABLE 10 Time Course for Mycelial Growthin Seed Culture Fermentation packed mycelium volume (pmv) % Fermentation(3000 dissolved period h pH rpms 5 min) oxygen % 0 5.7 ± 0.1 100 4 5.7 ±0.1 100 8 5.7 ± 0.1 12 ± 3 85 ± 5 12 5.7 ± 0.1 15 ± 3 72 ± 5 16 5.5 ±0.1 25 ± 5 40 ± 5 20 5.4 ± 0.1 30 ± 5 35 ± 5 22 5.3 ± 0.1 33 ± 5 30 ± 524 5.2 ± 0.1 35 ± 5 25 ± 5

[0414] Using the seed culture thus produced, a transformationfermentation run was carried out in a vertical cylindrical stainlesssteel fermenter having a diameter of 1.02 m, a height of 1.5 m and ageometric volume of 1.4 m³. The fermenter was provided with a turbineagitator having two impellers, one positioned 867 cm below the top ofthe reactor and the other positioned 1435 cm from the top. Each wheelwas provided with six blades, each 95 cm in radial dimension and 75 cmhigh. Vertical baffles 1440 cm high extended radially inwardly 100 cmfrom the interior vertical wall of the reactor. A nutrient mixture wasprepared having the composition set forth in Table 11 TABLE 11 ForBioconversion Culture in 1000 L Fermenter Quantity g/L glucose 16 kg 23peptone 16 kg 23 yeast autolysed 16 kg 23 antifoam SAG 471 0.080 Kgtraces deionized water q.s. to 700 L sterilize the empty fermenter for 1hour at 130° C. load it with 600 L ofdeionized water; heat at 40° C. addwhile stirring the components of the medium stir for 15 minutes, bringto volume of 650 L sterilization at 121° C. for 30 minutes poststerilization pH is 5.7 add sterile deionized water to 700 L

[0415] An initial charge (700 L) of this nutrient mixture (pH=5.7) wasintroduced into the fermenter, followed by the seed inoculum of thisexample (7 L) prepared as described above.

[0416] The nutrient mixture containing inoculum was incubated for 24hours at an aeration rate of 0.5 L/L-min at a head pressure of 0.5kg/cm². The temperature was controlled at 28° C., and the agitation ratewas 110 rpm. Growth of mycelium volume is shown in Table 12, along withpH and dissolved oxygen profiles of the seed culture reaction. TABLE 12Time Course for Mycelial Growth in Fermenter of the TransformationCulture packed mycelium volume (pmv) % Fermentation (3000 rpmx5dissolved period h pH min) oxygen % 0 5.6 ± 0.2 100 4 5.5 ± 0.2 100 85.5 ± 0.2 12 ± 3 95 ± 5 12 15 ± 3 90 ± 5 16 5.4 ± 0.1 20 ± 5 75 ± 5 205.3 ± 0.1 25 ± 5 60 ± 5 22 5.2 ± 0.1 30 ± 5 40 ± 5

[0417] At the conclusion of the incubation, pelleting of the myceliumwas observed, but the pellets were generally small and relativelyloosely packed. Diffuse mycelium was suspended in the broth. Final pHwas 5.1 to 5.3.

[0418] To the transformation culture thus produced was added asuspension of canrenone (1.250 kg; micronized to 5μ) in sterile water (5L). Sterile additive solution and antibiotic solution were added in theproportions indicated at reaction time 0 in Table 14. The composition ofthe additive solution is set forth in Table 13. TABLE 13 ADDITIVESOLUTION (for transformative culture) quantity dextrose 40 Kg yeastautolysate 8 Kg antifoam SAG 471 0.010 Kg deionized water q.s. to 100 lsterilize a 150 l empty fermenter for 1 hour at 130° C. load it with 70l of deionized water; heat at 40° C. add while stirring the componentsof “additive solution” stir for 30 minutes, bring to volume of 95 l pHas is 4.9 sterilize at 120° C. × 20 minutes pH after sterilization about5

[0419] Bioconversion was carried out for about 96 hours with aeration at0.5 L/L-min. at a head pressure of 0.5 kg/cm² and a pH of rangingbetween 4.7 and 5.3, adjusted as necessary by additions of 7.5 M NaOH or4 M H₃PO₄. The agitation rate was initially 100 rpm, increased to 165rpm at 40 hours and 250 rpm at 64 hours. The initial temperature was 28°C., lowered to 26° C. when PMV reached 45%, and lowered to 24° C. whenPMV rose to 60%. SAG 471 in fine drops was added as necessary to controlfoaming. Glucose levels in the fermentation were monitored at 4 hourintervals and, whenever the glucose concentration fell below 1 gpl, anincrement of sterile additive solution (10 L) was added to the batch.Disappearance of canrenone and appearance of 11α-hydroxycanrenone werealso monitored during the reaction by HPLC. When at least 90% of theinitial canrenone charge had been converted to 11α-hydroxycanrenone, anincrement of 1.250 kg canrenone was added. When 90% of the canrenone inthat increment was shown to have been converted, another 1.250 kgincrement was introduced. Using the same criterion further increments(1.250 kg apiece) were added until the total reactor charge (20 kg) hadbeen introduced. After the entire canrenone charge had been delivered tothe reactor, reaction was terminated when the concentration of unreactedcanrenone was 5% relative to the amount of 11α-hydroxycanrenoneproduced. The schedule for addition of canrenone, sterile additivesolution, and antibiotic solution is as shown in Table 14. TABLE 14Additions of the Steroid and Solutions (additives and antibiotics) inthe Course of Bioconversion of Canrenone in Fermenter CANRENONE Sterileanti- Reaction in suspension additive biotic volume time Progresssolution solution liters hours Kg -ive Kg liters liters about 0 1.2501.25 10 8 700 4 10 8 1.250 2.5 10 12 10 16 1.250 10 20 10 24 1.250 5 108 800 28 1.250 10 32 1.250 10 36 1.250 10 40 1.250 10 44 1.250 10 481.250 12.5 10 8 900 52 1.250 10 56 1.250 10 60 1.250 10 64 1.250 10 681.250 10 72 1.250 20 10 8 1050 76 0 80 84 88 92 Total

[0420] When bioconversion was complete, the mycelia were separated fromthe broth by centrifugation in a basket centrifuge. The filtrate wasdetermined by HPLC to contain only 2% of the total quantity of11α-hydroxycanrenone in the harvest broth, and was therefore eliminated.The mycelia were suspended in ethyl acetate (1000 L) in an extractiontank of 2 m³ capacity. This suspension was heated for one hour underagitation and ethyl acetate reflux conditions, then cooled andcentrifuged in a basket centrifuge. The mycelia cake was washed withethyl acetate (200 L) and thereafter discharged. The steroid richsolvent extract was allowed to stand for one hour for separation of thewater phase. The water phase was extracted with a further amount ofethyl acetate solvent (200 L) and then discarded. The combined solventphases were clarified by centrifugation and placed in a concentrator(500 L geometric volume) and concentrated under vacuum to a residualvolume of 100 L. In carrying out the evaporation, the initial charge tothe concentrator of combined extract and wash solutions was 100 L, andthis volume was kept constant by continual or periodic additions ofcombined solution as solvent was taken off. After the evaporation stepwas complete, the still bottoms were cooled to 20° C. and stirred fortwo hours, then filtered on a Buchner filter. The concentrator pot waswashed with ethyl acetate (20 L) and this wash solution was then used towash the cake on the filter. The product was dried under vacuum for 16hours at 50° C. Yield of 11α-hydroxycanrenone was 14 kg.

EXAMPLE 4

[0421] Lyophilized spores of Aspergillus ochraceus NRRL 405 weresuspended in a corn steep liquor growth medium (2 ml) having thecomposition set forth in Table 15: TABLE 15 Corn Steep Liquor Medium(Growth Medium for Primary Seed Cultivation) Corn steep liquor 30 gYeast extract 15 g Ammonium phosphate  3 g Monobasic Glucose (chargeafter sterilization) 30 g distilled water, q.s. to 1000 ml pH as is:4.6, adjust to pH 6.5 with 20% NaOH, distribute 50 ml to 250 mlErlenmeyer flask sterilize 121° C. for 20 minutes.

[0422] The resulting suspension was used in an inoculum for thepropagation of spores on agar plates. Ten agar plates were prepared,each bearing a solid glucose/yeast extract/phosphate/agar growth mediumhaving the composition set forth in Table 16: TABLE 16 GYPA(Glucose/Yeast Extract/Phosphate Agar for Plates) Glucose (charge aftersterilization) 10 g Yeast extract 2.5 g K₂HPO₄ 3 g Agar 20 g distilledwater, q.s. to 1000 ml adjust pH to 6.5 sterilize 121° C. for 30 minutes

[0423] A 0.2 ml aliquot of the suspension was transferred onto thesurface of each plate. The plates were incubated at 25° C. for ten days,after which the spores from all the plates were harvested into a sterilecryogenic protective medium having the composition set forth in Table17: TABLE 17 GYP/Glycerol (Glucose/Yeast Extract/ Phosphate/Glycerolmedium for stock vials) Glucose (charge after sterilization) 10 g Yeastextract 2.5 g K₂HPO₄ 3 g Glycerol 20 g Distilled water, q.s. to 1000 mLSterilize at 121° C. for 30 minutes

[0424] The resulting suspension was divided among twenty vials, with oneml being transferred to each vial. These vials constitute a master cellbank that can be drawn on to produce working cell banks for use ingeneration of inoculum for bioconversion of canrenone to11α-hydroxycanrenone. The vials comprising the master cell bank werestored in the vapor phase of a liquid nitrogen freezer at −130° C.

[0425] To begin preparation of a working cell bank, the spores from asingle master cell bank vial were resuspended in a sterile growth medium(1 ml) having the composition set forth in Table 15. This suspension wasdivided into ten 0.2 ml aliquots and each aliquot used to inoculate anagar plate bearing a solid growth medium having the composition setforth in Table 16. These plates were incubated for ten days at 25° C. Bythe third day of incubation, the underside of the growth medium wasbrown-orange. At the end of the incubation there was heavy production ofgolden colored spores. The spores from each plate were harvested by theprocedure described hereinabove for the preparation of the master cellbank. A total of one hundred vials was prepared, each containing 1 ml ofsuspension. These vials constituted the working cell bank. The workingcell bank vials were also preserved by storage in the vapor phase of aliquid nitrogen freezer at −130° C.

[0426] Growth medium (50 ml) having the composition set forth in Table15 was charged to a 250 ml Erlenmeyer flask. An aliquot (0.5 ml) ofworking cell suspension was introduced into the flask and mixed with thegrowth medium. The inoculated mixture was incubated for 24 hours at 25°C. to produce a primary seed culture having a percent packed mycelialvolume of approximately 45%. Upon visual inspection the culture wasfound to comprise pellet-like mycelia of 1 to 2 mm diameter; and uponmicroscopic observation it appeared as a pure culture.

[0427] Cultivation of a secondary seed culture was initiated byintroducing a growth medium having the composition set forth in Table 15into a 2.8 L Fernbach flask, and inoculating the medium with a portion(10 ml) of the primary seed culture of this example, the preparation ofwhich was as described above. The inoculated mixture was incubated at25° C. for 24 hours on a rotating shaker (200 rpm, 5 cm displacement).At the end of the incubation, the culture exhibited the same propertiesas described above for the primary seed culture, and was suitable foruse in a transformation fermentation in which canrenone was bioconvertedto 11α-hydroxycanrenone.

[0428] Transformation was conducted in a Braun E Biostat fermenterconfigured as follows: Capacity: 15 liters with round bottom Height: 53cm Diameter: 20 cm H/D: 2.65 Impellers: 7.46 cm diameter, six paddles2.2 × 1.4 cm each Impeller spacing: 65.5, 14.5 and 25.5 cm from bottomof tank Baffles: four 1.9 × 48 cm Sparger: 10.1 cm diameter, 21 holes ˜1mm diameter Temperature control: provided by means of an external vesseljacket

[0429] Canrenone at a concentration of 20 g/L was suspended in deionizedwater (4 L) and a portion (2 L) of growth medium having the compositionset forth in Table 18 was added while the mixture in the fermenter wasstirred at 300 rpm. TABLE 18 (Growth medium for bioconversion culture in10 L fermenter) Quantity Amount/L glucose (charge after 160 g 20 gsterilization) peptone 160 g 20 g yeast extract 160 g 20 g antifoamSAF471 4.0 ml 0.5 ml Canrenone 160 g 20 g deionized water q.s. to 7.5 Lsterilize 121° C. for 30 minutes

[0430] The resulting suspension was stirred for 15 minutes, after whichthe volume was brought up to 7.5 L with additional deionized water. Atthis point the pH of the suspension was adjusted from 5.2 to 6.5 byaddition of 20% by weight NaOH solution, and the suspension was thensterilized by heating at 121° C. for 30 minutes in the Braun Efermenter. The pH after sterilization was 6.3±0.2, and the final volumewas 7.0 L. The sterilized suspension was inoculated with a portion (0.5L) of the secondary seed culture of this example that has been preparedas described above, and the volume brought up to 8.0 L by addition of50% sterile glucose solution. Fermentation was carried out at atemperature of 28° C. until the PMV reached 50%, then lowered to 26° C.,and further lowered to 24° C. when PMV exceeded 50% in order to maintaina consistent PMV below about 60%. Air was introduced through the spargerat a rate of 0.5 vvm based on initial liquid volume and the pressure inthe fermenter was maintained at 700 millibar gauge. Agitation began at600 rpm and was increased stepwise to 1000 rpm as needed to keep thedissolved oxygen content above 30% by volume. Glucose concentration wasmonitored. After the initial high glucose concentration fell below 1%due to consumption by the fermentation reaction, supplemental glucosewas provided via a 50% by weight sterile glucose solution to maintainthe concentration in the 0.05% to 1% range throughout the remainder ofthe batch cycle. Prior to inoculation the pH was 6.3±0.2. After the pHdropped to about 5.3 during the initial fermentation period, it wasmaintained in the range of 5.5±0.2 for the remainder of the cycle byaddition of ammonium hydroxide. Foam was controlled by adding apolyethylene glycol antifoam agent sold under the trade designation SAG471 by OSI Specialties, Inc.

[0431] Growth of the culture took place primarily during the first 24hours of the cycle, at which time the PMV was about 40%, the pH wasabout 5.6 and the dissolved oxygen content was about 50% by volume.Canrenone conversion began even as the culture was growing.Concentrations of canrenone and 11α-hydroxycanrenone were monitoredduring the bioconversion by analyzing daily samples. Samples wereextracted with hot ethyl acetate and the resulting sample solutionanalyzed by TLC and HPLC. The bioconversion was deemed complete when theresidual canrenone concentration was about 10% of the initialconcentration. The approximate conversion time was 110 to 130 hours.

[0432] When bioconversion was complete, mycelial biomass was separatedfrom the broth by centrifugation. The supernatant was extracted with anequal volume of ethyl acetate, and the aqueous layer discarded. Themycelial fraction was resuspended in ethyl acetate using approximately65 volumes per g canrenone charged to the fermentation reactor. Themycelial suspension was refluxed for one hour under agitation, cooled toabout 20° C., and filtered on a Buchner funnel. The mycelial filter cakewas washed twice with 5 volumes of ethyl acetate per g of canrenonecharged to the fermenter, and then washed with deionized water (1 L) todisplace the residual ethyl acetate. The aqueous extract, rich solvent,solvent washing and water washing were combined. The remaining exhaustedmycelial cake was either discarded or extracted again, depending onanalysis for residual steroids therein. The combined liquid phases wereallowed to settle for two hours. Thereafter, the aqueous phase wasseparated and discarded, and the organic phase concentrated under vacuumuntil the residual volume was approximately 500 ml. The still bottle wasthen cooled to about 15° C. with slow agitation for about one hour. Thecrystalline product was recovered by filtration, and washed with chilledethyl acetate (100 ml). Solvent was removed from the crystals byevaporation, and the crystalline product dried under vacuum at 50° C.

EXAMPLE 5

[0433] Lyophilized spores of Aspergillus ochraceus ATCC 18500 weresuspended in a corn steep liquor growth medium (2 ml) as described inExample 4. Ten agar plates were prepared, also in the manner of Example4. The plates were incubated and harvested as described in Example 4 toprovide a master cell bank. The vials comprising the master cell bankwere stored in the vapor phase of a liquid nitrogen freezer at −130° C.

[0434] From a vial of the master cell bank, a working cell bank wasprepared as described in Example 4, and stored in the nitrogen freezerat −130° C.

[0435] Growth medium (300 mL) having the composition set forth in Table19 was charged to a 2 L baffled flask. An aliquot (3 mL) of working cellsuspension was introduced into the flask. The inoculated mixture wasincubated for 20 to 24 hours at 28° C. on a rotating shaker is (200 rpm,5 cm displacement) to produce a primary seed culture having a percentpacked mycelial volume of approximately 45%. Upon visual inspection theculture was found to comprise pellet like mycelia of 1 to 2 mm diameter;and upon microscopic observation it appeared as a pure culture. TABLE 19Growth medium for primary and secondary seed cultivation Amount/Lglucose (charge after 20 g sterilization) peptone 20 g Yeast extract 20g distilled water q.s. to 1000 mL sterilize 121° C. for 30 minutes

[0436] Cultivation of a secondary seed culture was initiated byintroducing 8 L growth medium having the composition set forth in Table19 into a 14 L glass fermenter. Inoculate the fermenter with 160 mL to200 mL of the primary seed culture of this example. The preparation ofwhich was as described above.

[0437] The inoculated mixture was cultivated at 28° C. for 18-20 hours,200 rmp agitation, aeration rate was 0.5 vvm. At the end of thepropagation, the culture exhibited the same properties as describedabove for the primary seed.

[0438] Transformation was conducted in a 60 L fermenter, substantiallyin the manner described in Example 4, except that the growth medium hadthe composition set forth in Table 20, and the initial charge ofsecondary seed culture was 350 mL to 700 mL. Agitation rate wasinitially 200 rpm, but increased to 500 rpm as necessary to maintaindissolved oxygen above 10% by volume. The approximate bioconversion timefor 20 g/L canrenone was 80 to 160 hours. TABLE 20 Growth Medium forBioconversion Culture in 60 L Fermenter Quantity Amount/L glucose(charge after 17.5 g 0.5 g sterilization) peptone 17.5 g 0.5 g yeastextract 17.5 g 0.5 g Canrenone (charge as a 700 g 20 g 20% slurry insterile water) deionized water, q.s. to 35 L sterilize 121° C. for 30minutes

EXAMPLE 6

[0439] Using a spore suspension from the working cell bank produced inaccordance with the method described in Example 4, primary and secondaryseed cultures were prepared, also substantially in the manner describedin Example 4. Using secondary seed culture produced in this manner, twobioconversion runs were made in accordance with a modified process ofthe type illustrated in FIG. 1, and two runs were made with the processillustrated in FIG. 2. The transformation growth medium, canrenoneaddition schedules, harvest times, and degrees of conversion for theseruns are set forth in Table 21. Run R2A used a canrenone addition schemebased on the same principle as Example 3, while run R2C modified theExample 3 scheme by making only two additions of canrenone, one at thebeginning of the batch, and one after 24 hours. In runs R2B and R2D, theentire canrenone charge was introduced at the beginning of the batch andthe process generally carried in the manner described in Example 4,except that the canrenone charge was sterilized in a separate vesselbefore it was charged to the fermenter and glucose was added as thebatch progressed. A Waring blender was used to reduce chunks produced onsterilization. In runs R2A and R2B, canrenone was introduced into thebatch in methanol solution, in which respect these runs further differedfrom the runs of Examples 3 and 4, respectively. TABLE 21 Descriptionsof the Initial Canrenone Bioconversion Processes Run Number R2A R2B R2CR2D Medium (g/L) Corn steep liq. 30 the same as run 30 the same as runYeast extract 15 R2A 15 R2C NH₄H₂PO₄  3  3 Glucose 15 30 OSA 0.5 ml 0.5ml pH adjusted to 6.0 adjusted to with 2.5 NNaOH 6.5 with 2.5 NNaOHCanrenone 10 g/80 ml MEOH 80 g/640 ml MEOH Sterilized and Sterilized andadded at 0, 18, added at 0 hr all blended; added blended; added 24, 30,36, 42, at once at: 0 hr: 25 g at: 0 hr: 200 g 50, 56, 62 and 24 hr: 200g 68 hr. Harvest time 143 hrs. 166 hrs. 125 hrs. 104 hrs. Bioconversion45.9% 95.6% 98.1% 95.1%

[0440] In runs R2A and R2B, the methanol concentration accumulated toabout 6.0% in the fermentation beer, which was found to be inhibitory tothe growth of culture and bioconversion. However, based on the resultsof these runs, it was concluded that methanol or other water-misciblesolvent could serve effectively at lower concentrations to increase thecanrenone charge and provide canrenone as a fine particle precipitateproviding a large interfacial area for supply of canrenone to thesubject to the reaction.

[0441] Canrenone proved stable at sterilization temperature (121° C.)but aggregated into chunks. A Waring blender was employed to crush thelumps into fine particles, which were successfully converted to product.

EXAMPLE 7

[0442] Using a spore suspension from the working cell bank produced inaccordance with the method described in Example 4, primary and secondaryseed cultures were prepared, also substantially in the manner describedin Example 4. The description and results of Example 7 are shown inTable 22. Using secondary seed culture produced in this manner, onebioconversion (R3C) was carried out substantially as described inExample 3, and three bioconversions were carried out in accordance withthe process generally described in Example 5. In the latter three runs(R3A, R3B and R3D), canrenone was sterilized in a portable tank,together with the growth medium except for glucose. Glucose wasaseptically fed from another tank. The sterilized canrenone suspensionwas introduced into the fermenter either before inoculation or duringthe early stage of bioconversion. In run R3B, supplemental sterilecanrenone and growth medium was introduced at 46.5. Lumps of canrenoneformed on sterilization were delumped through a Waring blender thusproducing a fine particulate suspension entering the fermenter. Thetransformation growth media, canrenone addition schedules, nutrientaddition schedules, harvest times, and degrees of conversion for theseruns are set forth in Tables 22 and 23. TABLE 22 Descriptions of Processof Canrenone Bioconversion Run Number R3A R3B R3C R3D Medium (g/L) Cornsteep hg. 30 the same as run Peptone: 20 the same as run Yeast extract15 R3A Yeast Ext.: 20 R3A NH₄H₂PO₄  3 Glucose: 20 Glucose 15 OSA: 3 mlOSA 0.5 ml pH adjusted to 6.5 adjusted to 6.5 with 2.5 N NaOH with 2.5NNaOH Canrenone canrenone was the same as run Non-sterile The same ascharge sterilized and R3A canrenone: run R3A at blended. BI: 50 g BI: 50g charged by the 16.5 hrs: 110 g 16.5 hrs: 110 g scheduled El: 50 g 46.5hrs: 80 g listed in 16.5 hrs: 110 g Table 23 Feedings see Table 23 seeTable 23 see Table 23 see Table 23 Harvest time 118.5 hrs. 118.5 hrs.118.5 hrs. 73.5 hrs. Bioconversion 93.7% 94.7% 60.0% 68.0%

[0443] TABLE 23 The Feeding Schedule for Canrenone, Glucose and GrowthMedium in the Development Experiment R3C R3A R3B R3D Peptone &Canrenone/ Canrenone/ Canrenone/ canrenone Yeast ext. Antibiotics GrowthGrowth Growth 200 g/2 L 20 g each 20 mg kanamycin Medium Medium Mediumsterile Glucose 50% in 1 L 20 mg tetracycline see see see Addition DIsolution water 100 mg cefalexin Table 22 Table 22 Table 22 Time hr. g gg in 50 ml g/L g/L g/L 0 — — — —  50 g/0.4 L  50 g/0.4 L  50 g/0.4 L14.5 16 100 25 50 ml — — — 16.5 — — — — 110 g/1.2 L 110 g/1.2 L 110g/1.2 L 20.5 16 140 25 — — — — 28.5 16 140 25 — — — — 34.5 16 150 25 — —— — 40.5 16 150 25 50 ml — — — 46.5 880 130 25 — — 80 g/0.8 L — 52.5 160120 25 — — — — 58.5 160 150 25 — — — — 64.5 160 180 25 50 ml — — — 70.5160 140 25 — — — —

[0444] Due to filamentous growth, a highly viscous fermenter broth wasseen in all four of the runs of this Example. To overcome obstacleswhich high viscosity created with respect to aeration, mixing, pHcontrol and temperature control, the aeration rate and agitation speedwere increased during these runs. Conversions proceeded satisfactorilyunder the more severe conditions, but a dense cake formed above theliquid broth surface. Some unreacted canrenone was carried out of thebroth by this cake.

EXAMPLE 8

[0445] The description and results of Example 8 are summarized in Table24. Four fermentation runs were made in which 11α-hydroxycanrenone wasproduced by bioconversion of canrenone. In two of these runs (R4A andR4D), the bioconversion was conducted in substantially the same manneras runs R3A and R3D of Example 6. In run R4C, canrenone was converted to11α-hydroxycanrenone generally in the manner described in Example 3. InRun R4B, the process was carried out generally as described in Example4, i.e., with sterilization of canrenone and growth medium in thefermenter just prior to inoculation, all nitrogen and phosphorusnutrients were introduced at the start of the batch, and a supplementalsolution containing glucose only was fed into the fermenter to maintainthe glucose level as the batch proceeded. In the latter process (runR4B), glucose concentration was monitored every 6 hours and glucosesolution added as indicated to control glucose levels in the 0.5 to 1%range. The canrenone addition schedules for these runs are set forth inTable 25. TABLE 24 Descriptions of the Process Development Experiment ofCanrenone Bioconversions Run Number R4A R4B R4C R4D Medium (g/L) Cornsteep liq. 30 the same as run Peptone: 20 the same as run Yeast extract15 R4A Yeast ext.: 20 R4A NH₄H₂PO₄  3 Glucose: 20 Glucose 15 OSA 3 mlOSA 0.5 ml pH adjusted to 6.5 adjusted to 6.5 with 2.5 NNaOH with 2.5NNaOH Canrenone Canrenone was 160 g canrenone is Nonsterile Canrenonewas charge at sterilized and sterilized in the canrenone: sterilized andblended. fermenter charged by the blended. BI: 40 g schedule listed BI:40 g 23.5 hrs: 120 g in Table 25 23.5 hrs: 120 g Medium charge see Table25 see Table 25 see Table 25 see Table 25 Harvest time 122 hrs. 122 hrs.122 hrs. 122 hrs. Bioconversion 95.6% 97.6% 95.4% 96.7%

[0446] TABLE 25 The Feeding Schedule of Canrenone, Glucose and GrowthMedium in the Development Experiment R4C Peptone & Antibiotics CanrenoneYeast ext. 20 mg kanamycin R4A R4B R4D 200 g/2 L Glucose 20 g each 20 mgtetracycline Growth Growth Growth sterile 50% in 1 L 100 mg cefalexinMedium Medium Medium Addition water solution water in 50 ml (added seesee see Time hr. g g g in canrenone slurry) Table 24 Table 24 Table 2414 600 135 25 50 ml — — — 20 — 100 — — — — — 23 — — — — 120 g/1.2 L —120 g/1.2 L 26 — 100 25 — — — — 32 — 135 25 — — — — 38 500 120 25 50 ml— — — 44 — 100 25 — — — — 50 — 100 25 — — — — 56 — 150 25 — — — — 62 500150 25 50 ml — — — 68 — 200 25 — — — — 74 — 300 25 — — — — 8- — 100 25 —— — — 86 — 125 25 — — — — 92 — 175 25 — — — — 98 — 150 — — — — — 104 —175 — — — — — 110 — 175 — — — — — 116 — 200 — — — — —

[0447] All fermenters were run under high agitation and aeration duringmost of the fermentation cycle because the fermentation beer had becomehighly viscous within a day or so after inoculation.

EXAMPLE 9

[0448] The transformation growth media, canrenone addition schedules,harvest times, and degrees of conversion for the runs of this Exampleare set forth in Table 26.

[0449] Four bioconversion runs were carried out substantially in themanner described for run R4B of Example 8, except as described below. Inrun R5B, the top turbine disk impeller used for agitation in the otherruns was replaced with a downward pumping marine impeller. The downwardpumping action axially poured the broth into the center of the fermenterand reduced cake formation. Methanol (200 ml) was added immediatelyafter inoculation in run R5D. Since canrenone was sterilized in thefermenter, all nutrients except glucose were added at the start of thebatch, obviating the need for chain feeding of sources of nitrogen,sources of phosphorus or antibiotics. TABLE 26 Process Description ofthe Process Development Experiment of 10 L Scale Bioconversions RunNumber R5A R5B R5C R5D Medium (g/L) Corn steep liq. 30 the same as runPeptone: 20 the same as run Yeast Extract 15 R5A Yeast Ext.: 20 R5ANH₄H₂PO₄  3 Glucose: 20 Glucose 15 OSA 3 ml OSA 0.5 ml pH adjusted to6.5 adjusted to 6.5 with 2.5 NNaOH with 2.5 NNaOH Canrenone 160 gcanrenone 160 g canrenone 160 g 160 g charge sterilized in thesterilized in the canrenone canrenone fermenter fermenter sterilized insterilized in the fermenter the fermenter Medium glucose feeding glucosefeeding glucose feeding glucose feeding feeding Harvest time 119.5 hrs.119.5 hrs. 106 119.5 hrs. Bioconversion 96% 94.1% 88.5% 92.4%

[0450] In order to maintain immersion of the solid phase growing abovethe liquid surface, growth medium (2 L) was added to each fermenter 96hours after the beginning of the batch. Mixing problems were notentirely overcome by either addition of growth medium or use of adownward pumping impeller (run R5B) but the results of the runsdemonstrated the feasibility and advantages of the process, andindicated that satisfactory mixing could be provided according toconventional practices.

EXAMPLE 10

[0451] Three bioconversion runs were carried out substantially in themanner described in Example 9. The transformation growth media,canrenone addition schedules, harvest times, and degrees of conversionfor the runs of this Example are set forth in Table 27: TABLE 27 ProcessDescription of the Experiment 10 L Scale Bioconversion Run Number R6AR6B R6C Medium (g/L) Corn steep liq. 30 the same as run Peptone: 20Yeast Extract 15 R6A Yeast Ext.: 20 NH₄H₂PO₄  3 Glucose: 20 Glucose 15OSA OSA 0.5 ml 0.5 ml pH adjusted to 6.5 adjusted to 6.5 with 2.5 N NaOHwith 2.5 N NaOH Canrenone 160 g canrenone 160 g canrenone 160 gcanrenone charge sterilized in the sterilized in the sterilized infermenter fermenter the fermenter Medium glucose feeding; glucosefeeding; glucose feeding 1.3 L medium 0.5 L medium feeding; no and 0.8 Lsterile and 0.5 L sterile other addition water at 71 hrs. water at 95hrs Harvest time 120 hrs. 120 hrs. 120 hrs. Bioconversion 95% 96% 90%Mass Balance 59% 54% 80%

[0452] Growth medium (1.3 L) and sterile water (0.8 L) were added after71 hours in run R⁶A to submerge mycelial cake which had grown above thesurface of the liquid broth. For the same purpose, growth medium (0.5 L)and sterile water (0.5 L) were added after 95 hours in run R⁶B. Materialbalance data showed that a better mass balance could be determined wherecake buildup above the liquid surface was minimized.

EXAMPLE 11

[0453] Fermentation runs were made to compare pre-sterilization ofcanrenone with sterilization of canrenone and growth medium in thetransformation fermenter. In run R7A, the process was carried out asillustrated in FIG. 2, under conditions comparable to those of runs R2C,R2D, R3A, R3B, R3D, R4A, and R4D. Run R7B was as illustrated in FIG. 3under conditions comparable to those of Examples 4, 9 and 10, and runR4B. The transformation growth media, canrenone addition schedules,harvest times, and degrees of conversion for the runs of this Exampleare set forth in Table 28: TABLE 28 Process Description of theExperiment of 10 L Scale Bioconversions Run Number R7A R7B Medium (g/L)corn steep liq. 30 the same as run Yeast extract 15 R7A NH₄H₂PO₄  3Glucose 15 OSA 0.5 ml pH adjusted to 6.5 with 2.5 NNaOH Canrenone 160 gcanrenone 160 g canrenone charge was sterilized & was sterilized blendedoutside in the fermenter the fermenter Medium charge Glucose feeding;Glucose feeding; canrenone was no other added with 1.6 L addition growthmedium Harvest time 118.5 hrs. 118.5 hrs. Bioconversion 93% 89%

[0454] A mass balance based on the final sample taken from run R⁷B was89.5%, indicating that no significant substrate loss or degradation inbioconversion. Mixing was determined to be adequate for both runs.

[0455] Residual glucose concentration was above the desired 5-10 gplcontrol range during the initial 80 hours. Run performance wasapparently unaffected by a light cake that accumulated in the head spaceof both the fermenters.

EXAMPLE 12

[0456] Extraction efficiency was determined in a series of 1 Lextraction runs as summarized in Table 29. In each of these runs,steroids were extracted from the mycelium using ethyl acetate (1 L/Lfermentation volume). Two sequential extractions were performed in eachrun. Based on RP-HPLC, About 80% of the total steroid was recovered inthe first extraction; and recovery was increased to 95% by the secondextraction. A third extraction would have recovered another 3% ofsteroid. The remaining 2% is lost in the supernatant aqueous phase. Theextract was drawn to dryness using vacuum but was not washed with anyadditional solvent. Chasing with solvent would improve recovery from theinitial extraction if justified by process economics. TABLE 29 Recoveryof 11α-Hydroxycanrenone at 1 Liter Extraction (% of Total) 1st 2nd 3rdRun Number Extract Extract Extract Supernatant R5A 79% 16% 2% 2% R5A 84%12% 2% 2% R4A 72% 20% 4% 4% R4A 79% 14% 2% 5% R4B 76% 19% 4% 1% R4B 79%16% 3% 2% R4B 82% 15% 2% 1% Average 79% 16% 3% 2%

[0457] Methyl isobutyl ketone (MIBK) and toluene were evaluated asextraction/crystallization solvents for 11α-hydroxycanrenone at the 1 Lbroth scale. Using the extraction protocol as described hereinabove,both MIBK and toluene were comparable to ethyl acetate in bothextraction efficiency and crystallization performance.

EXAMPLE 13

[0458] As part of the evaluation of the processes of FIGS. 2 and 3,particle size studies were conducted on the canrenone substrate providedat the start of the fermentation cycle in each of these processes. Asdescribed above, canrenone fed to the process of FIG. 1 was micronizedbefore introduction into the fermenter. In this process, the canrenoneis not sterilized, growth of unwanted microorganisms being controlled byaddition of antibiotics. The processes of FIGS. 2 and 3 sterilize thecanrenone before the reaction. In the process of FIG. 2, this isaccomplished in a blender before introduction of canrenone into thefermenter. In the process of FIG. 3, a suspension of canrenone in growthmedium is sterilized in the fermenter at the start of the batch. Asdiscussed hereinabove, sterilization tends to cause agglomeration ofcanrenone particles. Because of the limited solubility of canrenone inthe aqueous growth medium, the productivity of the process depends onmass transfer from the solid phase, and thus may be expected to dependon the interfacial area presented by the solid particulate substratewhich in turn depends on the particle size distribution. Theseconsiderations initially served as deterrents to the processes of FIGS.2 and 3.

[0459] However, agitation in the blender of FIG. 2 and the fermentationtank of FIG. 3, together with the action of the shear pump used fortransfer of the batch in FIG. 2, were found to degrade the agglomeratesto a particle size range reasonably approximate that of the unsterilizedand micronized canrenone fed to the process of FIG. 1. This isillustrated by the particle size distributions for the canrenone asavailable at the outset of the reaction cycle in each of the threeprocesses. See Table 30 and FIGS. 4 and 5. TABLE 30 ParticleDistributions of Three Different Canrenone Samples 45- mean Run #: %Sample 125 μ <180 μ size μ Bioconversion Canrenone 75% 95% — R3C:shipment 93.1% (120 h) R4C: 96.3% (120 h) Blended 31.2% 77.2% 139.5 R3A:Sample 94.6% (120 h) R3B: 95.2% (120 h) Sterilized 24.7% 65.1% 157.4R4B: Sample 97.6% (120 h) R5B: 93.8% (120 h)

[0460] From the data in Table 30, it will be noted that agitators andshear pump were effective to reduce the average particle size of thesterilized canrenone to the same order of magnitude as the unsterilizedsubstrate, but a significance size difference remained in favor of theunsterilized substrate. Despite this difference, reaction performancedata showed that the pre-sterilization processes were at least asproductive as the process of FIG. 1. Further advantages may be realizedin the process of FIG. 2 by certain steps for further reducing andcontrolling particle size, e.g., wet milling of sterilized canrenone,and/or by pasteurizing rather than sterilizing.

EXAMPLE 14

[0461] A seed culture was prepared in the manner described in Example 5.At 20 hours, the mycelia in the inoculum fermenter was pulpy with a 40%PMV. Its pH was 5.4 and 14.8 gpl glucose remained unused.

[0462] A transformation growth medium (35 L) was prepared having thecomposition shown in Table 20. In the preparation of feeding medium,glucose and yeast extract were sterilized separately and mixed as asingle feed at an initial concentration of 30% by weight glucose and 10%by weight yeast extract. pH of the feed was adjusted to 5.7.

[0463] Using this medium, (Table 20), two bioconversion runs were madefor the conversion of canrenone to 11α-hydroxycanrenone. Each of theruns was conducted in a 60 L fermenter provided with an agitatorcomprising one Rushton turbine impeller and two Lightnin' A315impellers.

[0464] Initial charge of the growth medium to the fermenter was 35 L.Micronized and unsterilized canrenone was added to an initialconcentration of 0.5%. The medium in the fermenter was inoculated with aseed culture prepared in the manner described in Example 5 at an initialinoculation ratio of 2.5%. Fermentation was carried out at a temperatureof 28° C., an agitation rate of 200 to 500 rpm, an aeration rate of 0.5vvm, and backpressure sufficient to maintain a dissolved oxygen level ofat least 20% by volume. The transformation culture developed during theproduction run was in the form of very small oval pellets (about 1-2mm). Canrenone and supplemental nutrients were chain fed to thefermenter generally in the manner described in Example 1. Nutrientadditions were made every four hours at a ratio of 3.4 g glucose and 0.6g yeast extract per liter of broth in the fermenter.

[0465] Set forth in Table 31 are the aeration rate, agitation rate,dissolved oxygen, PMV, and pH prevailing at stated intervals during eachof the runs of this Example, as well as the glucose additions madeduring the batch. Table 32 shows the canrenone conversion profile. RunR11A was terminated after 46 hours; Run R11B continued for 96 hours. Inthe latter run, 93% conversion was reached at 81 hours; one more feedaddition was made at 84 hours; and feeding then terminated. Note that asignificant change in viscosity occurred between the time feeding wasstopped and the end of the run. TABLE 31 PMV Gluc cc Time air (lpm) rpm% DO Backpress (%) pH (g/l) Fermentation R11A 0.1 20 200 93 0 2 6.17 5.87 20 200 85.1 0 5 6.03 5.5 12.4 20 300 50.2 0 5.43 21.8 20 400 25.5 0 386.98 0 29 20 500 17 0 35 5.22 30.2 20 500 18.8 10 5.01 31 20 500 79 104.81 1 35.7 20 500 100 10 45 5.57 0 46.2 20 500 23 6 45 5.8 1 Totalglucose: 27.5 g/l Total yeast extract: 8.75 g/l Fermentation R11B 0.1 20200 92.9 0 2 5.98 5.4 7 20 200 82.3 0 5 5.9 5 12.4 20 300 49.5 0 5.4821.8 20 400 18 0 40 7.12 0 29 20 500 36.8 0 35 5.1 3 35.7 20 500 94.5 104.74 0 46.2 20 500 14.5 6 45 5.32 2 55 20 500 16.7 10 5.31 0.5 58.6 20500 19.4 15 5.32 1 61.9 20 500 13 15 40 5.36 2 71.7 20 500 13 15 42 5.370 81.1 20 500 22.9 15 5.42 2.5 85.6 20 500 22 15 45 5.48 1 97.5 20 500108 15 45 6.47 0 17.7 20 500 15 7.38 0 Total glucose: 63 g/l Total yeastextract: 14.5 g/l

[0466] TABLE 32 Concentrations (g/l) Conversion Calc OH-can Conv. rates(g/l/h) Sample Time OH-can Canren. Total (%) (g/l) Calculated MeasuredFermentation R11A: Canrenone conversion R11A-0 0.10 0.00 5.41 5.41R11A-7 7.00 0.18 4.89 5.07 3.58 0.18 0.03 0.03 R11A- 21.80 2.02 2.124.14 48.75 2.44 0.15 0.12 22 R11A- 29.00 3.67 4.14 7.81 47.03 4.48 0.280.23 29 R11A- 35.70 6.68 1.44 8.12 82.27 7.74 0.49 0.45 36 R11A- 46.207.09 0.41 7.51 94.48 8.59 0.08 0.04 46 Fermentation R11B: Canrenoneconversion R11B-0 0.1 0.00 5.60 5.60 R11B-7 7.0 0.20 4.98 5.18 3.78 0.190.03 0.03 R11B- 21.8 2.51 2.46 4.97 50.49 2.52 0.16 0.16 22 R11B- 29.04.48 16.99 21.47 20.87 4.69 0.30 0.27 29 R11B- 35.7 8.18 10.35 18.5344.16 9.70 0.75 0.55 36 R11B- 55.0 17.03 13.20 30.23 56.33 19.50 0.320.36 55 R11B- 58.6 20.80 11.73 32.53 63.95 21.97 0.69 1.05 59 R11B- 61.922.19 8.62 30.81 72.02 24.50 0.77 0.42 62 R11B- 71.7 26.62 3.61 30.2388.06 29.46 0.51 0.45 72 R11B- 81.1 27.13 2.05 29.18 92.97 30.32 0.090.05 81 R11B- 85.6 26.87 2.02 28.88 93.02 30.11 −0.04 −0.06 86 R11B-97.5 23.95 1.71 25.66 93.34 30.22 0.01 −0.25 97 R11B- 117.7 24.10 1.6825.79 93.47 30.26 0.00 0.01 118

EXAMPLE 15

[0467] Various cultures were tested for effectiveness in thebioconversion of canrenone to 11α-canrenone according to the methodsgenerally described above.

[0468] A working cell bank of each of Aspergillus niqer ATCC 11394,Rhizopus arrhizus ATCC 11145 and Rhizopus stolonifer ATCC 6227b wasprepared in the manner described in Example 5. Growth medium (50 ml)having the composition set forth in Table 18 was inoculated with asuspension of spores (1 ml) from the working cell bank and placed in anincubator. A seed culture was prepared in the incubator by fermentationat 26° C. for about 20 hours. The incubator was agitated at a rate of200 rpm.

[0469] Aliquots (2 ml) of the seed culture of each microorganism wereused to inoculate transformation flasks containing the growth medium (30ml) of Table 18. Each culture was used for inoculation of two flasks, atotal of six. Canrenone (200 mg) was dissolved in methanol (4 ml) at 36°C., and a 0.5 ml aliquot of this solution was introduced into each ofthe flasks. Bioconversion was carried out generally under the conditionsdescribed in Example 5 with additions of 50% by weight glucose solution(1 ml) each day. After the first 72 hours the following observationswere made on the development of mycelia in the respective transformationfermentation flasks:

[0470] ATCC 11394—good even growth

[0471] ATCC 11145—good growth in first 48 hours, but mycelial clumpedinto a ball; no apparent growth in last 24 hours;

[0472] ATCC 6227b—good growth; mycelial mass forming clumped ball.

[0473] Samples of the broth were taken to analyze for the extent ofbioconversion. After three days, the fermentation using ATCC 11394provided conversion to 11α-hydroxycanrenone of 80 to 90%; ATCC 11145provided a conversion of 50%; and ATCC 6227b provided a conversion of 80to 90%.

EXAMPLE 16

[0474] Using the substantially the method described in Example 15, theadditional microorganisms were tested for effectiveness in theconversion of canrenone to 11α-hydroxycanrenone. The organisms testedand the results of the tests are set forth in Table 33: TABLE 33Cultures tested for Bioconversion of canrenone to 11alpha-hydroxy-canrenone approximate Culture ATTC # media¹ resultsconversion Rhizopus oryzae 1145 CSL + 50% — Rhizopus stolonifer 6227bCSL + 80-90% — Aspergillus nidulans 11267 CSL + 50%  80% Aspergillusniger 11394 CSL + 80-90% — Aspergillus ochraceus NRRL 405 CSL +  90%Aspergillus ochraceus 18500 CSL +  90% Bacillus subtilis 31028 P & CSL − 0%  0% Bacillus subtilis 31028 CSL −  0%  0% Bacillus sp. 31029 P & CSL−  0%  0% Bacillus sp. 31029 CSL −  0% * Bacillus megaterium 14945 P &CSL +  5%  80%* Bacillus megaterium 14945 CSL +  5%  10%* Trichotheciumroseum 12519 CSL + 80%*  90%* Trichothecium roseum 8685 CSL + 80%*  90%*Streptomyces fradiae 10745 CSL + <5% <10% Streptomyces fradiae 10745 TSB− * * Streptomyces 13664 CSL −  0% * lavendulae Streptomyces 13664 TSB − 0%  0% lavendulae Nocardiodes simplex 6946 BP −  0%  0% Nocardiodessimplex 13260 BP − * * Pseudomonas sp. 14696 BP − * * Pseudomonas sp.14696 CSL + <5% <10% Pseudomonas sp. 14696 TSB −  0% * Pseudomonas sp.13261 BP + * <10% Pseudomonas cruciviae 13262 BP # <10% Pseudomonasputida 15175 BP −  0%  0%

EXAMPLE 17

[0475] Various microorganisms were tested for effectiveness in theconversion of canrenone to 9α-hydroxycanrenone. Fermentation media forthe runs of this Example were prepared as set forth in Table 34: TABLE34 Soybean Meal: dextrose 20 g soybean meal 5 g NaCl 5 g yeast extract 5g KH₂PO₄ 5 g water to 1 L pH 7.0 Peptone/yeast extract/glucose: glucose40 g bactopeptone 10 g yeast extract 5 g water to 1 L Mueller-Hinton:beef infusion 300 g casamino acids 17.5 g starch 1.5 g water to 1 L

[0476] Fungi were grown in soybean meal medium and in peptone-yeastextract glucose; atinomycetes and eubacteria were grown in soybean meal(plus 0.9% by weight Na formate for biotransformations) and inMueller-Hinton broth.

[0477] Starter cultures were inoculated with frozen spore stocks (20 mlsoybean meal in 250 ml Erlenmayer flask). The flasks were covered with amilk filter and bioshield. Starter cultures (24 or 48 hours old) wereused to inoculate metabolism cultures (also 20 ml in 250 ml Erlenmeyerflask)—with a 10% to 15% crossing volume—and the latter incubated for 24to 48 hours before addition of steroid substrate for the transformationreaction.

[0478] Canrenone was dissolved/suspended in methanol (20 mg/ml), filtersterilized, and added to the cultures to a final concentration of 0.1mg/ml. All transformation fermentation flasks were shaken at 250 rpm(21″ throw) in a controlled temperature room at 26° C. and 60% humidity.

[0479] Biotransformations were harvested at 5 and 48 hours, or at 24hours, after addition of substrate. Harvesting began with the additionof ethyl acetate (23 ml) or methylene chloride to the fermentationflask. The flasks were then shaken for two minutes and the contents ofeach flask poured into a 50 ml conical tube. To separate the phases,tubes were centrifuged at 4000 rpm for 20 minutes in a room temperatureunit. The organic layer from each tube was transferred to a 20 mlborosilicate glass vial and evaporated in a speed vac. Vials were cappedand stored at −20° C.

[0480] To obtain material for structure determination,biotransformations were scaled up to 500 ml by increasing the number ofshake flask fermentations to 25. At the time of harvest (24 or 48 hoursafter addition of substrate), ethyl acetate was added to each flaskindividually, and the flasks were capped and put back on the shaker for20 minutes. The contents of the flasks were then poured intopolypropylene bottles and centrifuged to separate the phases, or into aseparatory funnel in which phases were allowed to separate by gravity.The organic phase was dried, yielding crude extract of steroidscontained in the reaction mixture.

[0481] Reaction product was analyzed first by thin layer chromatographyon silica gel (250 μm) fluorescence backed plates (254 nm). Ethylacetate (500 μL was added to each vial containing dried ethyl acetateextract from the reaction mixture.Further analyses were conducted byhigh performance liquid chromatography and mass spectrometry. Plateswere developed in a 95:5 v/v chloroform/methanol medium.

[0482] Further analysis was conducted by high performance liquidchromatography and mass spectrometry. A waters HPLC with Millenniumsoftware, photodiode array detector and autosampler was used. Reversedphase HPLC used a waters NovaPak C-18 (4 μm particle size) RadialPak 4mmcartridge. The 25 minute linear solvent gradient began with the columninitialized in water:acetonitrile (75:25), and ended atwater:acetonitrile (25:75). This was followed by a three minute gradientto 100% acetonitrile and 4 minutes of isocratic wash before columnregeneration in initial conditions.

[0483] For LC/MS, ammonium acetate was added to both the acetonitrileand water phases at a concentration of 2 nM. Chromatography was notsignificantly affected. Eluant from the column was split 22:1, with themajority of the material directed to-the PDA detector. The remaining4.5% of the material was directed to the electrospray ionizing chamberof an Sciex API III mass spectrometer. Mass spectrometry wasaccomplished in positive mode. An analog data line from the PDA detectoron the HPLC transferred a single wave length chromatogram to the massspectrometer for coanalysis of the UV and MS data.

[0484] Mass spectrometric fragmentation patterns proved useful insorting from among the hydroxylated substrates. The two expectedhydroxylated canrenones, 11α-hydroxy- and 9α-hydroxy, lost water atdifferent frequencies in a consistent manner which could be used as adiagnostic. Also, the 9α-hydroxycanrenone formed an ammonium adduct morereadily than did 11α-hydroxycanrenone. Set forth in Table 35 is asummary of the TLC, HPLC/UV and LC/MS data for canrenone fermentations,showing which of the tested microorganism were effective in thebioconversion of canrenone to 9α-hydroxycanrenone. Of, these, thepreferred microorganism was Corynespora cassiicola ATCC 16718. TABLE 35Summary of TLC, HPLC/UV, and LC/MS Data for Canrenone FermentationsEvidence for 9αOH-canrenone MS: 357 HPLC-peak (M + H), TLC spot at 9αOH-339(−H₂O) at 9αQH- canrenone & 375 Culture AD w/UV (+NH₄) Absidiacoerula ATCC n y y/n 6647 Absidia glauca ATCC n 22752 Actinomucorelegans ATCC tr y tr 6476 Aspergillus flavipes tr ATCC 1030 Aspergillusfumigatus tr y n ATCC 26934 Aspergillus nidulans tr y v ATCC 11267Aspergillus niger ATCC n y y 16888 Aspergillus niger ATCC n y n 26693Aspergillus ochraceus n y n ATCC 18500 Bacterium cyclo-oxydans n tr n(Searle) ATCC 12673 Beauveria bassiana ATCC tr y y 7159 Beauveriabassiana ATCC y y y 13144 Botrvosphaeria obtusa y tr tr IMI 038560Calonectria decora ATCC n tr y 14767 Chaetomium cochliodes tr tr y/nATCC 10195 Comomonas testosteroni tr tr n (Searle) ATCC 11996Corynespora cassiicola y y y ATCC 16718 Cunninghamella y y y blakesleanaATCC 8688a Cunninghamella y y y echinulata ATCC 3655 Cunninghamellaelegans y y y ATCC 9245 Curcularia clavata ATCC n y y/n 22921 Curvularialunata ATCC y n n 12071 Cylindrocarpora tr n n radicicola (Searle) ATCC11011 Epicoccum humucola ATCC y y y 12722 Epicoccum orvzae ATCC tr tr tr12724 Fusarium oxysporum ATCC tr 7601 Fusarium oxysporum f.sp. n cepaeATCC 11171 Gibberella fujikurci tr y y ATCC 14842 Gliocladiumdeliquescens y tr tr ATCC 10097 Gongronella butieri ATCC y y UV ? y22822 Hypomyces chrysospermus y y y Tul. IMI 109891 Lipomyces lipoferATCC n 10792 Melanospora ornata ATCC tr n n 26180 Mortierellaisabellinay y y n ATCC 42613 Mucor grisco-cyaraus ATCC n 1207a Mucormucedo ATCC 4605 tr y y Mycobacterium fortuitumn ATCC 6842 Myrotheciumverrucaria tr tr y ATCC 9095 Nocardia aurentia n tr n (Searle) ATCC12674 Nocardia cancicruria y y n (Searle) Nocardia corallina ATCC n19070 Paecilomyces carneus n y n ATCC 46579 Penicillium chrysogenum nATCC 9480 Penicillium Datulum ATCC y y y/n 24550 Penicilliumpurvurogenum tr y y ATCC 46581 Pithomyces atro- tr y tr olivaceus ATCC6651 Pithomyces cynodontis n tr tr ATCC 26150 Phycomyces blakesleeanus yy Pycnosporium sp. ATCC y y y/n 12231 Rhizopogon sp. Rhizopus arrhizusATCC tr y n 11145 Rhizopus stolonifer ATCC n 6227b Rhodococcus equi ATCCn tr n 14887 Rhodococcus equi ATCC tr tr n 21329 Rhodococcus sp. n n nRhodococcus rhodochrous n tr n ATCC 19150 Saccharopolyspora y y yerythaea ATCC 11635 Sepedonium ampullosporum n n n IMI 203033 Sepedoniumchrysospermum n ATCC 13378 Settomvxa affinis ATCC n y UV? y/n 6737Stachylidium bicolor y y y/n ATCC 12672 Streptomyces n califorraicusATCC 15436 Streptomyces n cinereocrocatus ATCC 3443 Streptomycescoelicolor n ATCC 10147 Streptomyces flocculus ATCC 25453 Streptomycesfradiae n ATCC 10745 Streptomyces griseus n subsp. griseus ATCC 13968Streptomyces griseus n ATCC 11984 Streptomyces hydrogerians n ATCC 19631Streptomyces y y y hygroscopicus ATCC 27438 Streptomyces lavendulae nPanlab 105 Streptomyces n paucisporogenes ATCC 25489 Streptomyces n trtr purpurascens ATCC 25489 Streptomyces roseochromogenes ATCC 13400Streptomyces spectabilis n ATCC 27465 Stysanus microsporus ATCC 2833Syncephalastrum n racemosum ATCC 18192 Thaninidium elegans ATCC 18191Thamnostylum piriforme y tr y ATCC 8992 Thielavia terricolan ATCC 13807Trichoderma viride ATCC n 26802 Trichothecium roseum tr y y/n ATCC 12543Verticillium theobromae y tr tr ATCC 12474

EXAMPLE 18

[0485] Various cultures were tested for effectiveness in thebioconversion of androstendione to 11α-hydroxyandrostendione accordingto the methods generally described above.

[0486] A working cell bank of each of Aspergillus ochraceus NRRL 405(ATCC 18500); Aspergillus niger ATCC 11394; Aspergillus nidulans ATCC11267; Rhizopus oryzae ATCC 11145; Rhizopus stolonifer ATCC 6227b;Trichothecium roseum ATCC 12519 and ATCC 8685 was prepared essentiallyin the manner described in Example 4. Growth medium (50 ml) having thecomposition set forth in Table 18 was inoculated with a suspension ofspores (1 ml) from the working cell bank and placed in an incubator. Aseed culture was prepared in the incubator by fermentation at 26° C. forabout 20 hours. The incubator was agitated at a rate of 200 rpm.

[0487] Aliquots (2 ml) of the seed culture of each microorganism wereused to inoculate transformation flasks containing the growth medium (30ml) of Table 15. Each culture was used for inoculation of two flasks, atotal of 16. Androstendione (300 mg) was dissolved in methanol (6 ml) at36° C., and a 0.5 ml aliquot of this solution was introduced into eachof the flasks. Bioconversion was carried out generally under theconditions described in Example 6 for 48 hours. After 48 hours samplesof the broth were pooled and extracted with ethyl acetate as in Example17. The ethyl acetate was concentrated by evaporation, and samples wereanalyzed by thin layer chromatography to determine whether a producthaving a chromatographic mobility similar to that of11α-hydroxy-androstendione standard (Sigma Chemical Co., St. Louis) waspresent. The results are shown in Table 36. Positive results areindicated as “+”. TABLE 36 Bioconversion of androstendione to 11 alpha-hydroxy-andro St endi one TLC Culture ATTC# media results Rhizopusoryzae 11145 CSL + Rhizopus stolonifer 6227b CSL + Aspergillus nidulans11267 CSL + Aspergillus niger 11394 CSL + Aspergillus ochraceus NRRL 405CSL + Aspergillus ochraceus 18500 CSL + Trichothecium roseum 12519 CSL +Trichothecium roseum 8685 CSL +

[0488] The data in Table 36 demonstrate that each of listed cultures wascapable of producing a compound from androstendione having the same Rfvalue as that of the 11α-hydroxyandrostendione standard.

[0489]Aspergillus ochraceus NRRL 405 (ATCC 18500) was retested by thesame procedure described above, and the culture products were isolatedand purified by normal phase silica gel column chromatography usingmethanol as the solvent. Fractions were analyzed by thin layerchromatography. TLC plates were Whatman K6F silica gel 60 Å, 10×20 size,250μ thickness. The solvent system was methanol:chloroform, 5:95, v/v.The crystallized product and 11α-hydroxyandrostendione standard wereboth analyzed by LC-MS and NMR spectroscopy. Both compounds yieldedsimilar profiles and molecular weights.

EXAMPLE 19

[0490] Various microorganisms were tested for effectiveness in theconversion of mexrenone to 11β-hydroxymexrenone. Fermentation media forthis example were prepared as described in Table 34.

[0491] The fermentation conditions and analytical methods were the sameas those in Example 17. TLC plates and the solvent system were asdescribed in Example 18. The rationale for chromatographic analysis isas follows: 11α-hydroxymexrenone and 11α-hydroxycanrenone have the samechromatographic mobility. 11α-hydroxycanrenone and 9α-hydroxycanrenoneexhibit the same mobility pattern as 11α-hydroxyandrostendione and11β-hydroxyandrostendione. Therefore, 11β-hydroxymexrenone should havethe same mobility as 9α-hydroxycanrenone. Therefore, compounds extractedfrom the growth media were run against 9α-hydroxycanrenone as astandard. The results are shown in Table 36. TABLE 37 Summary of TLCData for 11β- hydroxymexrenone Formation from Mexrenone SpotMicroorganism Medium¹ Character² Absidia coerula ATCC 6647 M, S strongAspergillus niger ATCC S, P faint (S) 16888 ? (P) Beauveria bassianaATCC P strong 7159 Beauveria bassiana ATCC S,P ?, ? 13144 Botryosphaeriaobtusa IMI faint 038560 Cunninghamella blakesleeana ATCC 8688a S, Pstrong echinulata ATCC 3655 S, P strong elegans ATCC 9245 S, P strongCurvularia lunata ATCC S strong 12017 Gongronella butleri ATCC S, Pstrong 22822 Penicillium Datulum ATCC S, P strong 24550 Penicilliumpurpurogenum S, P strong ATCC 46581 Pithomyces atro-olivaceus S, P faintIFO 6651 Rhodococcus equi ATCC M faint 14887 Saccharopolyspora erythaeaM, SF faint ATCC 11635 Streotomyces hygroscopicus M, SF strong ATCC27438 Streptomyces purpurascens M, SF faint ATCC 25489 Thamnidiumelegans ATCC S, P faint 18191 Thamnostylum piriforme S, P faint ATCC8992 Trichothecium roseum ATCC P, S faint (P) 12543 ? (S)

[0492] These data suggest that the majority of the organisms listed inthis table produce a product similar or identical to11β-hydroxymexrenone from mexrenone.

EXAMPLE 20

[0493] Scheme 1: Step 1: Preparation of5′R(5′α),7′β-20′-Aminohexadecahydro-11′β-hydroxy-10′a,13′β-dimethyl-3′,5-dioxospiro[furan-2(3H),17′α(5′H)-[7,4]metheno[4H]cyclopenta[a]phenanthrene]-5′-carbonitrile.

[0494] Into a 50 gallon glass-line reactor was charged 61.2 L (57.8 kg)of DMF followed by 23.5 Kg of 11-hydroxycanrenone 1 with stirring. Tothe mixture was added 7.1 kg of lithium chloride. The mixture wasstirred for 20 minutes and 16.9 kg of acetone cyanohydrin was chargedfollowed by 5.1 kg of triethylamine. The mixture was heated to 85° C.and maintained at this temperature for 13-18 hours. After the reaction353 L of water was added followed by 5.6 kg of sodium bicarbonate. Themixture was cooled to 0° C., transferred to a 200 gallon glass-linedreactor with quenched with 130 kg of 6.7% sodium hypochlorite solutionslowly. The product was filtered and washed with 3×40 L portions ofwater to give 21.4 kg of the product enamine.

EXAMPLE 21

[0495] Scheme 1: Step 2: Preparation of4′S(4′α),7′α-Hexadecahydro-11′α-hydroxy-10′β,13′β-dimethyl-3′,5,20′-trioxospiro[furan-2(3H),17′β-[4,7]methano[17H]cyclopenta[a]phenanthrene]-5′β(2′H)-carbonitrile.

[0496] Into a 200 gallon glass-lined reactor was charged 50 kg ofenamine 2, approximately 445 L of 0.8 N dilute hydrochloric acid and 75L of methanol. The mixture was heated to 80° C. for 5 hours, cooled to0° C. for 2 hours. The solid product was filtered to give 36.5 kg of dryproduct diketone.

[0497] Scheme 1: Step 3A: Preparation of Methyl Hydrogen11α,17α-Dihydroxy-3-oxopregn-4-ene-7α,21-dicarboxylate, γ-Lactone.

[0498] A 4-neck 5-L bottom flask was equipped with mechanical stirrer,pressure equalizing addition funnel with nitrogen inlet tube,thermometer and condenser with bubbler. The bubbler was connected viatygon tubing to two 2-L traps, the first of which was empty and placedto prevent back-suction of the material in the second trap (1 L ofconcentrated sodium hypochlorite solution) into the reaction vessel. Thediketone 3 (79.50 g; [weight not corrected for purity, which was 85%])was added to the flask in 3 L methanol. A 25% methanolic sodiummethoxide solution (64.83 g) was placed in the funnel and addeddropwise, with stirring under nitrogen, over a 10 minute period. Afterthe addition was complete, the orangish yellow reaction mixture washeated to reflux for 20 hours. After this period, 167 mL of 4 N HCl wasadded (Caution: HCN evolution at this point!) dropwise through theaddition funnel to the still refluxing reaction mixture. The reactionmixture lightened in color to a pale golden orange. The condenser wasthen replaced with a take-off head and 1.5 L of methanol was removed bydistillation while 1.5 L of water was simultaneously added to the flaskthrough the funnel, in concert with the distillation rate. The reactionmixture was cooled to ambient temperature and extracted twice with 2.25L aliquots of methylene chloride. The combined extracts were washedsuccessively with 750 mL aliquots of cold saturated NaCl solution, 1NNaOH and again with saturated NaCl. The organic layer was dried oversodium sulfate overnight, filtered and reduced in volume to ˜250 mL invacuo. Toluene (300 mL) was added and the remaining methylene chloridewas stripped under reduced pressure, during which time the product beganto form on the walls of the flask as a white solid. The contents of theflask were cooled overnight and the solid was removed by filtration. Itwas washed with 250 mL toluene and twice with 250 mL aliquots of etherand dried on a vacuum funnel to give 58.49 g of white solid was 97.3%pure by HPLC. On concentrating the mother liquor, an additional 6.76 gof 77.1% pure product was obtained. The total yield, adjusted forpurity, was 78%.

EXAMPLE 23

[0499] Scheme 1: Step 3B: Conversion of Methyl Hydrogen11α,17α-Dihydroxy-3-oxopregn-4-ene-7α,21-dicarboxylate, γ-Lactone toMethyl Hydrogen17α-Hydroxy-11α-(methylsulfonyl)oxy-3-oxopregn-4-ene-7α,21-dicarboxylate,γ-Lactone.

[0500] A 5-L four neck flask was equipped as in the above example,except that no trapping system was installed beyond the bubbler. Aquantity of 138.70 g of the hydroxyester was added to the flask,followed by 1425 mL methylene chloride, with stirring under nitrogen.The reaction mixture was cooled to −5° C. using a salt/ice bath.Methanesulfonyl chloride (51.15 g, 0.447 mole) was added rapidly,followed by the slow dropwise addition of triethylamine (54.37 g) in 225mL methylene chloride. Addition, which required ˜30 minutes, wasadjusted so that the temperature of the reaction never rose about 5° C.Stirring was continued for 1 hour post-addition, and the reactioncontents were transferred to a 12-L separatory funnel, to which wasadded 2100 mL methylene chloride. The solution was washed successivelywith 700 mL aliquots each of cold 1N HCl, 1N NaOH, and saturated aqueousNaCl solution. The aqueous washes were combined and back-extracted with3500 mL methylene chloride. All of the organic washes were combined in a9-L jug, to which was added 500 g neutral alumina, activity grade II,and 500 g anhydrous sodium sulfate. The contents of the jug were mixedwell for 30 minutes and filtered. The filtrate was taken to dryness invacuo to give a gummy yellow foam. This was dissolved in 350 mLmethylene chloride and 1800 mL ether was added dropwise with stirring.The rate of addition was adjusted so that about one-half of the etherwas added over 30 minutes. After about 750 mL had been added, theproduct began to separate as a crystalline solid. The remaining etherwas added in 10 minutes. The solid was removed by filtration, and thefilter cake was washed with 2 L of ether and dried in a vacuum oven at50° C. overnight, to give 144.61 g (88%) nearly white solid, m.p.149-150° C. Material prepared in this fashion is typically 98-99% pureby HPLC (area %). In one run, material having a melting point of153°-153.5° C. was obtained, with a purity, as determined by HPLC area,of 99.5%.

EXAMPLE 24

[0501] Scheme 1: Step 3C: Method A: Preparation of Methyl Hydrogen17α-Hydroxy-3-oxopregna-4,9(11)-diene-7α,21-dicarboxylate, γ-Lactone.

[0502] A 1-L four neck flask was equipped as in the second example.Formic acid (250 mL) and acetic anhydride (62 mL) were added to theflask with stirring under nitrogen. Potassium formate (6.17 g) was addedand the reaction mixture was heated with an oil bath to an internaltemperature of 40° C. (this was later repeated at 70° C with betterresults) for 16 hours. After 16 hours, the mesylate 5 was added and theinternal temperature was increased to 100° C. Heating and stirring werecontinued for 2 hours, after which the solvent was removed in vacuo on arotavap. The residue was stirred with 500 mL ice water for fifteenminutes, then extracted twice with 500 mL aliquots of ethyl acetate. Theorganic phases were combined and washed successively with cold 250 mLaliquots of saturated sodium chloride solution (two times), 1 N sodiumhydroxide solution, and again with saturated sodium chloride. Theorganic phase was then dried over sodium sulfate, filtered and taken todryness in vacuo to give a yellowish white foam, which pulverized to aglass when touched with a spatula. The powder that formed, 14.65 ganalyzed as a mixture of 82.1% 6 7.4% 8 and 5.7% 9 (by HPLC area %).

EXAMPLE 25

[0503] Scheme 1: Step 3C: Method B: Preparation of Methyl Hydrogen17α-Hydroxy-3-oxopregna-4,9(11)-diene-7α,21-dicarboxylate, γ-Lactone.

[0504] A 5-L four neck flask was equipped as in the above example and228.26 g acetic acid and 41.37 g sodium acetate were added with stirringunder nitrogen. Using an oil bath, the mixture was heated to an internaltemperature of 100° C. The mesylate (123.65 g) was added, and heatingwas continued for thirty minutes. At the end of this period, heating wasstopped and 200 mL of ice water was added. The temperature dropped to40° C. and stirring was continued for 1 hour, after which the reactionmixture was poured slowly into 1.5 L of cold water in a 5-L stirredflask. The product separated as a gummy oil. The oil was dissolved in 1L ethyl acetate and washed with 1 L each cold saturated sodium chloridesolution, 1 N sodium hydroxide, and finally saturated sodium chlorideagain. The organic phase was dried over sodium sulfate and filtered. Thefiltrate was taken to dryness in vacuo to give a foam which collapsed toa gummy oil. This was triturated with ether for some time and eventuallysolidified. The solid was filtered and washed with more ether to afford79.59 g of a yellow white solid. This consisted of 70.4% of the desiredΔ^(9,11) enester 6, 12.3% of the Δ^(11,12) enester 8, 10.8% of the 7-α,9-α-lactone 9 and 5.7% unreacted 5.

EXAMPLE 26

[0505] Scheme 1: Step 3D: Synthesis of Methyl Hydrogen9,11α-Epoxy-17α-hydroxy-3-oxopregn-4-ene-7α,21-dicarboxylate, γ-Lactone.

[0506] A 4-neck jacketed 500 mL reactor was equipped with mechanicalstirrer, condenser/bubbler, thermometer and addition funnel withnitrogen inlet tube. The reactor was charged with 8.32 g of the crudeenester in 83 mL methylene chloride, with stirring under nitrogen. Tothis was added 4.02 g dibasic potassium phosphate, followed by 12 mL oftrichloroacetonitrile. External cooling water was run through thereactor jacket and the reaction mixture was cooled to 8° C. To theaddition funnel 36 mL of 30% hydrogen peroxide was added over a 10minute period. The initially pale yellow colored reaction mixture turnedalmost colorless after the addition was complete. The reaction mixtureremained at 9±1° C. throughout the addition and on continued stirringovernight (23 hours total). Methylene chloride (150 mL) was added to thereaction mixture and the entire contents were added to ˜250 mL icewater. This was extracted three times with 150 mL aliquots of methylenechloride. The combined methylene chloride extracts were washed with 400mL cold 3% sodium sulfite solution to decompose any residual peroxide.This was followed by a 330 mL cold 1 N sodium hydroxide wash, a 400 mLcold 1 N hydrochloric acid wash, and finally a wash with 400 mL brine.The organic phase was dried over magnesium sulfate, filtered, and thefilter cake was washed with 80 mL methylene chloride. Solvent wasremoved in vacuo to give 9.10 g crude product as a pale yellow solid.This was recrystallized from ˜25 mL 2-butanone to give 5.52 g nearlywhite crystals. A final recrystallization from acetone (˜50 mL gave 3.16g long, acicular crystals, mp 241-243° C.

EXAMPLE 27

[0507] Scheme 1: Step 3: Option 1: From 4′S(4′α),7′α-Hexadecahydro-11′α-hydroxy-10′β,13′β-dimethyl-3′,5,20′-trioxospiro[furan-2(3H),17′β-[4,7]methano[17H]cyclopenta[a]phenanthrene]-5′β(2′H)-carbonitrileto Methyl Hydrogen 9,11α-Epoxy-17α-hydroxy-3-oxopregn-4-ene-7α,21-dicarboxylate, γ-Lactone.

[0508] Diketone (20 g) was charged into a clean and dried reactorfollowed by the addition of 820 ml of MeOH and 17.6 ml of 25% NaOMe/MeOHsolution. The reaction mixture was heated to reflux condition (˜67° C.)for 16-20 hours. The product was quenched with 40 mL of 4N HCl. Thesolvent was removed at atmospheric pressure by distillation. 100 mL oftoluene was added and the residual methanol was removed by azeotropedistillation with toluene. After concentration, the crude hydroxyester 4was dissolved in 206 mL of methylene chloride and cooled to 0° C.Methanesulfonyl chloride (5 mL) was added followed by a slow addition of10.8 ml of triethylamine. The product was stirred for 45 minutes. Thesolvent was removed by vacuum distillation to give the crude mesylate 5.

[0509] In a separate dried reactor was added 5.93 g of potassiumformate, 240 mL of formic acid and followed by 118 mL of aceticanhydride. The mixture was heated to 70° C. for 4 hours.

[0510] The formic acid mixture was added to the concentrated mesylatesolution 5 prepared above. The mixture was heated to 95-105° C. for 2hours. The product mixture was cooled to 50° C. and the volatilecomponents were removed by vacuum distillations at 50° C. The productwas partitioned between 275 ml of ethyl acetate and 275 ml of water. Theaqueous layer was back extracted with 137 ml of ethyl acetate, washedwith 240 ml of cold 1N sodium hydroxide solution and then 120 ml ofsaturated NaCl. After phase separation, the organic layer wasconcentrated to under vacuum distillation to give crude enester.

[0511] The product was dissolved in 180 mL of methylene chloride andcooled to 0 to 15° C. 8.68 g of dipotassium hydrogen phosphate was addedfollowed by 2.9 mL of trichloroacetonitrile. A 78 mL solution of 30%hydrogen peroxide was added to the mixture over a 3 minute period. Thereaction mixture was stirred at 0-15° C. for 6-24 hours. After thereaction, the two phase mixture was separated. The organic layer waswashed with 126 mL of 3% sodium sulfite solution, 126 mL of 0.5 N sodiumhydroxide solution, 126 mL of 1 N hydrochloric acid and 126 mL of 10%brine. The product was dried over anhydrous magnesium sulfate orfiltered over Celite and the solvent methylene chloride was removed bydistillation at atmospheric pressure. The product was crystallized frommethylethyl ketone twice to give 7.2 g of eplerenone.

EXAMPLE 28

[0512] Scheme 1: Step 3: Option 2: Conversion ◯1′S(4′α),7′α-Hexadecahydro-11′α-hydroxy-10′β,13′β-dimethyl-3′,5,20′-trioxospiro[furan-2(3H),17′β-[4,7]methano[17H]cyclopenta[a]phenanthrene]-5′β(2′H)-carbonitrileto Methyl Hydrogen9,11α-Epoxy-17α-hydroxy-3-oxopregn-4-ene-7α,21-dicarboxylate, γ-Lactonewithout intermediate.

[0513] A 4-neck 5-L round bottom flask was equipped with mechanicalstirrer, addition funnel with nitrogen inlet tube, thermometer andcondenser with bubbler attached to a sodium hypochlorite scrubber. Thediketone (83.20 g) was added to the flask in 3.05 L methanol. Theaddition funnel was charged with 67.85 g of a 25% (w:w) solution ofsodium methoxide in methanol. With stirring under nitrogen, themethoxide was added dropwise to the flask over a 15 minute period. Adark orange/yellow slurry developed. The reaction mixture was heated toreflux for 20 hours and 175 mL 4 N hydrochloric acid was added dropwisewhile refluxing continued. (Caution, HCN evolution during thisoperation!) The reflux condenser was replaced with a takeoff head and1.6 L of methanol was removed by distillation while 1.6 L of aqueous 10%sodium chloride solution was added dropwise through the funnel, at arate to match the distillation rate. The reaction mixture was cooled toambient temperature and extracted twice with 2.25 L of aliquots ofmethylene chloride. The combined extracts were washed with cold 750 mLaliquots of 1 N sodium hydroxide and saturated sodium chloride solution.The organic layer was dried by azeotropic distillation of the methanolat one atmosphere, to a final volume of 1 L (0.5% of the total wasremoved for analysis).

[0514] The concentrated organic solution (hydroxyester) was added backto the original reaction flask equipped as before, but without the HCNtrap. The flask was cooled to 0° C. and 30.7 g methanesulfonyl chloridewas added with stirring under nitrogen. The addition funnel was chargedwith 32.65 g triethylamine, which was added dropwise over a 15 minuteperiod, keeping the temperature at 5° C. Stirring was continued for 2hours, while the reaction mixture warmed to ambient. A column consistingof 250 g Dowex 50 Wx8-100 acid ion exchange resin was prepared and waswashed before using with 250 mL water, 250 mL methanol and 500 mLmethylene chloride. The reaction mixture was run down this column andcollected. A fresh column was prepared and the above process wasrepeated. A third 250 g column, consisting of Dowex 1x8-200 basic ionexchange resin was prepared and pretreated as in the acid resintreatment described above. The reaction mixture was run down this columnand collected. A fourth column of the basic resin was prepared and thereaction mixture again was run down the column and collected. Eachcolumn pass was followed by two 250 mL methylene chloride washes downthe column, and each pass required ˜10 minutes. The solvent washes werecombined with the reaction mixture and the volume was reduced in vacuoto ˜500 mL and 2% of this was removed for qc. The remainder was furtherreduced to a final volume of 150 mL (crude mesylate solution).

[0515] To the original 5-L reaction set-up was added 960 mL formic acid,472 mL acetic anhydride and 23.70 g potassium formate. This mixture washeated with stirring under nitrogen to 70° C. for 16 hours. Thetemperature was then increased to 100° C. and the crude mesylatesolution was added over a thirty minute period via the addition funnel.The temperature dropped to 85° C. as methylene chloride was distillingout of the reaction mixture. After all of it had been removed, thetemperature climbed back to 100° C., and was held there for 2.5 hours.The reaction mixture was cooled to 40° C. and the formic acid wasremoved under pressure until the minimum stir volume had been reached(˜150 mL). The residue was cooled to ambient and 375 mL methylenechloride was added. The diluted residue was washed with cold 1 Lportions of saturated sodium chloride solution, 1 N sodium carbonate,and again with sodium chloride solution. The organic phase was driedover magnesium sulfate (150 g), and filtered to give a dark reddishbrown solution (crude enester solution).

[0516] A 4-neck jacketed 1 L reactor was equipped with mechanicalstirrer, condenser/bubbler, thermometer and addition funnel withnitrogen inlet tube. The reactor was charged with the crude enestersolution (estimated 60 g) in 600 mL methylene chloride, with stirringunder nitrogen. To this was added 24.0 g dibasic potassium phosphate,followed by 87 mL trichloroacetonitrile. External cooling water was runthrough the reactor jacket and the reaction mixture was cooled to 10° C.To the addition funnel 147 mL 30% hydrogen peroxide was added mixtureover a 30 minute period. The initially dark reddish brown coloredreaction mixture turned a pale yellow after the addition was complete.The reaction mixture remained at 10±1° C. throughout the addition and oncontinued stirring overnight (23 hours total). The phases were separatedand the aqueous portion was extracted twice with 120 mL portions ofmethylene chloride. The combined organic phases were then washed with210 mL 3% sodium sulfite solution was added. This was repeated a secondtime, after which both the organic and aqueous parts were negative forperoxide by starch/iodide test paper. The organic phase was successivelywashed with 210 mL aliquots of cold 1 N sodium hydroxide, 1 Nhydrochloric acid, and finally two washes with brine. The organic phasewas dried azeotropically to a volume of ˜100 mL, fresh solvent was added(250 mL and distilled azeotropically to the same 100 mL and theremaining solvent was removed in vacuo to give 57.05 g crude product asa gummy yellow foam. A portion (51.01 g) was further dried to a constantweight of 44.3 g and quantitatively analyzed by HPLC. It assayed at27.1% EPX.

EXAMPLE 29

[0517] 11α-Hydroxyandrostendione (429.5 g) and toluene sulfonic acidhydrate (7.1) were charged to a reaction flask under nitrogen. Ethanol(2.58 L) was added to the reactor, and the resulting solution cooled to5° C. Triethyl orthoformate (334.5 g) was added to the solution over a15 minute period at 0° to 15° C. After the triethyl orthoformateaddition was complete the reaction mixture was warmed to 40° C. andreacted at that temperature for 2 hours, after which the temperature wasincreased to reflux and reaction continued under reflux for anadditional 3 hours. The reaction mixture was cooled under vacuum and thesolvent removed under vacuum to yield 3-ethoxyandrosta-3,5-diene-17-one.

EXAMPLE 30 Formation of Enamine from 11α-hydroxycanrenone

[0518]

[0519] Sodium cyanide (1.72 g) was placed in 25 mL 3-neck flask fittedwith a mechanical stirrer. Water (2.1 mL) was added and the mixture wasstirred with heating until the solids dissolved. Dimethylformamide (15mL) was added followed by 11α-hydroxycanrenone (5.0 g). A mixture ofwater (0.4 mL) and sulfuric acid (1.49 g) was added to mixture. Themixture was heated to 85° C. for 2.5 hours at which time HPLC analysisshowed complete conversion to product. The reaction mixture was cooledto room temperature. Sulfuric acid (0.83 g) was added and the mixturestirred for one half hour. The reaction mixture was added to 60 mL watercooled in an ice bath. The flask was washed with 3 mL DMF and 5 mLwater. The slurry was stirred for 40 min. and filtered. The filter cakewas washed twice with 40 mL water and dried in a vacuum oven at 60° C.overnight to yield the 11α-hydroxy enamine, i.e.,5′R(5′α),7′β-20′-aminohexadecahydro-11′β-hydroxy-10′α,13′α-dimethyl-3′,5-dioxospiro[furan -2(3H),17′α(5′H)-[7,4]metheno[4H] cyclopenta[a]phenanthrene]-5′-carbonitrile (4.9 g).

EXAMPLE 31 Conversion of 11α-hydroxycanrenone to Diketone

[0520]

[0521] Sodium cyanide (1.03 g) was added to a 50 mL 3-neck flask fittedwith a mechanical stirrer. Water (1.26 mL) was added and the flask washeated slightly to dissolve the solid. Dimethylacetamide [ordimethyformamide] (9 mL) was added followed by 11α-hydroxycanrenone (3.0g). A mixture of sulfuric acid (0.47 mL) and water (0.25 mL) was addedto the reaction flask while stirring. The mixture was heated to 95° C.for 2 hours. HPLC analysis indicated that the reaction was complete.Sulfuric acid (0.27 mL) was added and the mixture stirred for 30 min.Additional water (25 mL) and sulfuric acid (0.90 mL) were introduced andthe reaction mixture stirred for 16 hours. The mixture was then cooledin an ice bath to 5-10° C. The solid was isolated by filtering through asintered glass filter followed by washing twice with water (20 mL). Thesolid diketone, i.e.,4′S(4′α),7′α-Hexadecahydro-11′α-hydroxy-10′β,13′β-dimethyl-3′,5,20′-trioxospiro[furan-2(3H),17′β-[4,7]methano[17H]cyclopenta[a]phenanthrene]-5′β(2′H)-carbonitrilewas dried in a vacuum oven to yield 3.0 g of a solid.

EXAMPLE 32

[0522] A suspension of 5.0 g of the diketone produced in the mannerdescribed in Example 31 in methanol (100 mL) was heated to reflux and a25% solution of potassium methoxide in methanol (5.8 mL) was added over1 min. The mixture became homogeneous. After 15 min., a precipitate waspresent. The mixture was heated at reflux and again became homogeneousafter about 4 hours. Heating at reflux was continued for a total of 23.5hours and 4.0 N HCl (10 mL) was added. A total of 60 mL of a solution ofhydrogen cyanide in methanol was removed by distillation. Water (57 mL)as added to the distillation residue over 15 min. The temperature of thesolution was raised to 81.5° during water addition and an additional 4mL of hydrogen cyanide/methanol solution was removed by distillation.After water addition was complete, the mixture became cloudy and theheat source was removed. The mixture was stirred for 3.5 hours andproduct slowly crystallized. The suspension was filtered and thecollected solid was washed with water, dried in a stream of air on thefunnel, and dried at 92° (26 in. Hg) for 16 hours to give 2.98 g of anoff-white solid. The solid was 91.4% of the hydroxyester, i.e., methylhydrogen 11α,17α-dihydroxy-3-oxopregn-4-ene-7α,21-dicarboxylate,γ-lactone by weight. The yield was 56.1%.

EXAMPLE 33

[0523] Diketone prepared in the manner described in Example 31 wascharged into a cleaned and dried 3-neck reaction flask equipped with athermometer, a Dean Stark trap and a mechanical stirrer. Methanol (24mL) was charged to the reactor at room temperature (22° C.) and theresulting slurry stirred for 5 min. A 25% by weight solution of sodiummethoxide in methanol (52.8 mL) was charged to the reactor and themixture stirred for 10 min. at room temperature during which thereaction mixture turned to a light brown clear solution and a slightexotherm was observed (2-3° C.). The addition rate was controlled toprevent the pot temperature from exceeding 30° C. The mixture wasthereafter heated to reflux conditions (about 67° C.) and continuedunder reflux for 16 hrs. A sample was then taken and analyzed by HPLCfor conversion. The reaction was continued under reflux until theresidual diketone was not greater than 3% of the diketone charge. Duringreflux 4 N HCl (120 mL) was charged to the reaction pot resulting in thegeneration of HCN which was quenched in a scrubber.

[0524] After conclusion of the reaction, 90-95% of the methanol solventwas distilled out of the reaction mixture at atmospheric pressure. Headtemperature during distillation varied from 67-75° C. and the distillatewhich contained HCN was treated with caustic and bleach before disposal.After removal of methanol the reaction mixture was cooled to roomtemperature, solid product beginning to precipitate as the mixturecooled in the 40-45° C. range. An aqueous solution containing optionally5% by weight sodium bicarbonate (1200 mL) at 25° C. was charged to thecooled slurry and the resultant mixture then cooled to 0° C. in about 1hr. Sodium bicarbonate treatment was effective to eliminate residualunreacted diketone from the reaction mixture. The slurry was stirred at0° C. for 2 hrs. to complete the precipitation and crystallization afterwhich the solid product was recovered by filtration and the filter cakewashed with water (100 mL). The product was dried at 80-90° C. under 26″mercury vacuum to constant weight. Water content after drying was lessthan 0.25% by weight. Adjusted molar yield was around 77-80% by weight.

EXAMPLE 34

[0525] Diketone as prepared in accordance with Example 31 (1 eq.) wasreacted with sodium methoxide (4.8 eqs.) in a methanol solvent in thepresence of zinc iodide (1 eq.). Work up of the reaction product can beeither in accordance with the extractive process described herein, or bya non-extractive process in which methylene chloride extractions, brineand caustic washes, and sodium sulfate drying steps are eliminated. Alsoin the non-extractive process, toluene was replaced with 5% by weightsodium bicarbonate solution.

EXAMPLE 35

[0526] The hydroxyester prepared as by Example 34 (1.97 g) was combinedwith tetrahydrofuran (20 mL) and the resulting mixture cooled to −70° C.Sulfuryl chloride (0.8 mL) was added and the mixture was stirred for 30min., after which imidazole (1.3 g) was added. The reaction mixture waswarmed to room temperature and stirred for an additional 2 hrs. Themixture was then diluted with methylene chloride and extracted withwater. The organic layer was concentrated to yield crude enester (1.97g). A small sample of the crude product was analyzed by HPLC. Theanalysis showed that the ratio of 9,11-olefin:11,12-olefin:7,9-lactonewas 75.5:7.2:17.3. When carried out at 0° C. but otherwise as describedabove, the reaction yielded a product in which the9,11-olefin:11,12-olefin:7,9-lactone distribution was 77.6:6.7:15.7.This procedure combines into one step the introduction of a leavinggroup and elimination thereof for the introduction of the 9,11-olefinstructure of the enester, i.e., reaction was sulfuryl chloride causesthe 11α-hydroxy group of the hydroxy ester of Formula V to be replacedby halide and this is followed by dehydrohalogenation to the Δ-9,11structure. Thus formation of the enester is effected without the use ofa strong acid (such as formic) or a drying agent such as aceticanhydride. Also eliminated is the refluxing step of the alternativeprocess which generates carbon monoxide.

EXAMPLE 36

[0527] Hydroxyester (20 g) prepared as by Example 34, and methylenechloride (400 mL) were added to a clean dry three-neck round bottomflask fitted with a mechanical stirrer, addition funnel andthermocouple. The resulting mixture was stirred at ambient temperatureuntil complete solution was obtained. The solution was cooled to 5° C.using an ice bath. Methanesulfonyl chloride (5 mL) was added to thesolution of CH₂Cl₂ containing the hydroxyester, rapidly followed by theslow dropwise addition of triethylamine (10.8 mL). The addition rate wasadjusted so that the temperature of the reaction did not exceed 5° C.The reaction was very exothermic; therefore cooling was necessary. Thereaction mixture was stirred at about 5° C. for 1 h. When the reactionwas complete (HPLC and TLC analysis), the mixture was concentrated atabout 0° C. under 26 in Hg vacuum until it became a thick slurry. Theresulting slurry was diluted with CH₂Cl₂ (160 mL), and the mixture wasconcentrated at about 0° C. under 26 in Hg vacuum to obtain aconcentrate. The purity of the concentrate (mesylate product of FormulaIV wherein R³═H and —A—A— and —B—B— are both —CH₂—CH₂—, i.e., methylhydrogen 11α,17α-dihydroxy-3-oxopregn-4-ene-7α,21-dicarboxylate,γ-lactone to methyl hydrogen17α-hydroxy-11α-(methylsulfonyl)oxy-3-oxopregn-4-ene-7α,21-dicarboxylate,γ-lactone was found to be 82% (HPLC area %). This material was used forthe next reaction without isolation.

[0528] Potassium formate (4.7 g), formic acid (16 mL) and aceticanhydride (8 mL, 0.084 mol) were added to a clean dry reactor equippedwith mechanical stirrer, condenser, thermocouple and heating mantle. Theresulting solution was heated to 70° C. and stirred for about 4-8 hours.The addition of acetic anhydride is exothermic and generated gas (CO),so that the rate of addition had to be adjusted to control bothtemperature and gas generation (pressure). The reaction time to preparethe active eliminating reagent was dependent on the amount of waterpresent in the reaction (formic acid and potassium formate containedabout 3-5% water each). The elimination reaction is sensitive to theamount of water present; if there is >0.1% water (KF), the level of the7,9-lactone impurity may be increased. This by product is difficult toremove from the final product. When the KF showed <0.1% water, theactive eliminating agent was transferred to the concentrate of mesylate(0.070 mol) prepared in the previous step. The resulting solution washeated to 95° C. and the volatile material was distilled off andcollected in a Dean Stark trap. When volatile material evolution ceased,the Dean Stark trap was replaced with the condenser and the reactionmixture was heated for additional 1 h at 95° C. Upon completion (TLC andHPLC analysis; <0.1% starting material) the content was cooled to 50° C.and vacuum distillation was started (26 in Hg/50° C.). The mixture wasconcentrated to a thick slurry and then cooled to ambient temperature.The resulting slurry was diluted with ethyl acetate (137 mL) and thesolution was stirred for 15 min. and diluted with water (137 mL). Thelayers were separated, and the aqueous lower layer was re-extracted withethyl acetate (70 mL). The combined ethyl acetate solution was washedonce with brine solution (120 mL) and twice with ice cold lN NaOHsolution (120 mL each). The pH of aqueous was measured, and the organiclayer rewashed if the pH of the spent wash liquor was <8. When the pH ofthe spent wash was observed to be >8, the ethyl acetate layer was washedonce with brine solution (120 mL) and concentrated to dryness by rotaryevaporation using a 50° C. water bath. The resulting enester, solidproduct i.e., methyl hydrogen17α-hydroxy-3-oxopregna-4,9(11)-diene-7α,21-dicarboxylate, γ-lactoneweighed 92 g (77% mol yield).

EXAMPLE 37

[0529] Hydroxyester (100 g; 0.22 mol) prepared as by Example 34 wascharged to a 2 L 3-neck round bottom flask equipped with mechanicalstirrer, addition funnel, and thermocouple. A circulating cooling bathwas used with automatic temperature control. The flask was dried priorto reaction because of the sensitivity of methanesulfonyl chloride towater.

[0530] Methylene chloride (1 L) was charged to the flask and thehydroxyester dissolved therein under agitation. The solution was cooledto 0° C. and methane sulfonyl chloride (25 mL; 0.32 mol) was charged tothe flask via the addition funnel. Triethylamine (50 mL; 0.59 mol) wascharged to the reactor via the addition funnel and the funnel was rinsedwith additional methylene chloride (34 mL). Addition of triethylaminewas highly exothermic. Addition time was around 10 min. under agitationand cooling. The charge mixture was cooled to 0° C. and held at thattemperature under agitation for an additional 45 min. during which thehead space of the reaction flask was flushed with nitrogen. A sample ofthe reaction mixture was then analyzed by thin layer chromatography andhigh performance liquid chromatography to check for reaction completion.The mixture was thereafter stirred at 0° C. for an additional 30 min.and checked again for reaction completion. Analysis showed the reactionto be substantially complete at this point; the solvent methylenechloride was stripped at 0° C. under 26″ mercury vacuum. Gaschromatography analysis of the distillate indicated the presence of bothmethane sulfonyl chloride and triethylamine. Methylene chloride (800 mL)was thereafter charged to the reactor and the resulting mixture wasstirred for 5 min. at a temperature in the range of 0-15° C. The solventwas again stripped at 0-5° C. under 26″ mercury vacuum yielding themesylate of Formula IV wherein R³ is H, —A—A— and —B—B— are —CH₂—CH₂—and R¹ is methoxy carbonyl. The purity of the product was about 90-95area %.

[0531] To prepare an elimination reagent, potassium formate (23.5 g;0.28 mol), formic acid (80 mL) and acetic anhydride (40 mL) were mixedin a separate dried reactor. Formic acid and acetic anhydride werepumped into the reactor and the temperature was maintained not greaterthan 40° C. during addition of acetic anhydride. The elimination reagentmixture was heated to 70° C. to scavenge water from the reaction system.This reaction was continued until the water content was lower than 0.3%by weight as measured by Karl Fisher analysis. The elimination reagentsolution was then transferred to the reactor containing the concentratedcrude mesylate solution prepared as described above. The resultingmixture was heated to a maximum temperature of 95° C. and volatiledistillate collected until no further distillate was generated.Distillation ceased at about 90° C. After distillation was complete, thereaction mixture was stirred at 95° C. for an additional 2 hrs. andcompletion of the reaction was checked for thin layer chromatography.When the reaction was complete, the reactor was cooled to 50° C. and theformic acid and solvent removed from the reaction mixture under 26″mercury vacuum at 50° C. The concentrate was cooled to room temperatureand thereafter ethyl acetate (688 mL) was introduced and the mixture ofethyl acetate and concentrate stirred for 15 min. At this point, a 12%brine solution (688 mL) was introduced to assist in removing watersoluble impurities from the organic phase. The phases were then allowedto settle for 20 min. The aqueous layer was transferred to anothervessel to which an additional amount of ethyl acetate (350 mL) wascharged. This back extraction of the aqueous layer was carried out for30 min. after which the phases were allowed to settle and the ethylacetate layers combined. To the combined ethyl acetate layers, saturatedsodium chloride solution (600 mL) was charged and stirring carried outfor 30 min. The phases were then allowed to settle. The aqueous layerwas removed. An additional sodium chloride (600 mL) wash was carriedout. The organic phase was separated from the second spent wash liquor.The organic phase was then washed with 1 N sodium hydroxide (600 mL)under stirring for 30 min. The phases were settled for 30 min. to removethe aqueous layer. The pH of the aqueous layer was checked and it foundto be >7. A further wash was carried out with saturated sodium chloride(600 mL) for 15 min. The organic phase was finally concentrated under26″ mercury vacuum at 50° C. and the product recovered by filtration.The final product was a foamy brown solid when dried. Further drying at45° C. under reduced pressure for 24 hrs. yielded 95.4 g of the enesterproduct which assayed at 68.8%. The molar yield was 74.4% corrected forboth the starting hydroxy ester and the final enester.

EXAMPLE 38

[0532] The procedure of Example 37 was repeated except that the multiplewashing steps were avoided by treating the reaction solution with an ionexchange resin. Basic alumina or basic silica. Conditions for treatmentwith basic silica are set forth in Table 38. Each of these treatmentswas found effective for removal of impurities without the multiplewashes of Example 44. TABLE 38 Factor Set point Purpose of ExperimentKey results Basic 2 g/125 g Treating the reaction mixture The yieldalumina product with basic alumina to remove was 93% Et₃N.HCl salt andto eliminate the 1N NaOH and 1N HCl washes Basic 2 g/125 g Treating thereaction mixture The yield silica product with basic silica which is was95% cheaper to remove Et₃N.HCl salt and eliminate 1N NaOH and 1N HClwashes

EXAMPLE 39

[0533] Potassium acetate (4 g) and trifluoroacetic acid (42.5 mL) weremixed in a 100 mL reactor. trifluoroacetic anhydride (9.5 mL) was addedto the mixture at a rate controlled to maintain temperature duringaddition below 30° C. The solution was then heated to 30° C. for 30 min.to provide an elimination reagent useful for converting the mesylate ofFormula IV to the enester of Formula II.

[0534] The preformed TFA/TFA anhydride elimination reagent was added toa previously prepared solution of the mesylate of Formula IV. Theresulting mixture was heated at 40° C. for 4½ hrs., the degree ofconversion being periodically checked by TLC or HPLC. When the reactionwas complete, the mixture was transferred to 1-neck flask andconcentrated to dryness under reduced pressure at room temperature (22°C.). Ethyl acetate (137 mL) was added to the mixture to obtain completedissolution of solid phase material after which a water/brine mixture(137 mL) was added and the resulting two phase mixture stirred for 10min. The phases were then allowed to separate for 20 min. Brine strengthwas 24% by weight. The aqueous phase was contacted with an additionalamount of ethyl acetate (68 mL) and the two phase mixture thus preparedwas stirred for 10 min. after which it was allowed to stand for 15 min.for phase separation. The ethyl acetate layers from the two extractionswere combined and washed with 24% by weight brine (120 mL), anotheraliquot of 24% by weight brine (60 mL), 1 N sodium hydroxide solution(150 mL) and another portion of brine (60 mL). After each aqueous phaseaddition, the mixture was stirred for 10 min. and allowed to stand for15 min. for separation. The resulting solution was concentrated todryness under reduced pressure at 45° C. using a water aspirator. Thesolid product (8.09 g) was analyzed by HPLC and found to include 83.4area % of the enester, 2.45 area % of the 11,12-olefin, 1.5% of the7,9-lactone, and 1.1% of unreacted mesylate.

EXAMPLE 40

[0535] The mesylate having the structure prepared per Example 23 (1.0g), isopropenyl acetate (10 g) and p-toluenesulfonic acid (5 mg) wereplaced in a 50 ml flask and heated to 90° C. with stirring. After 5hours the mixture was cooled to 25° C. and concentrated in vacuo at 10mm of Hg. The residue was dissolved in CH₂Cl₂ (20 ml) and washed with 5%aqueous NaHCO₃. The CH₂Cl₂ layer was concentrated in vacuo to give 1.47g of a tan oil. This material was recrystallized from CH₂Cl₂/Et₂O togive 0.50 g of enol acetate of Formula IV(Z).

[0536] This material was added to a mixture of sodium acetate (0.12 g)and acetic acid (2.0 ml) that had been previously heated to 100° C. withstirring. After 60 minutes the mixture was cooled to 25° C. and dilutedwith CH₂Cl₂ (20 ml). The solution was washed with water (20 ml) anddried over MgSO₄. The drying agent was removed by filtration and thefiltrate was concentrated in vacuo to give 0.4 g of the desired9,11-olefin, IV(Y). The crude product contained less than 2% of the7,9-lactone impurity.

EXAMPLE 41 Thermo Elimination of Mesylate in DMSO

[0537]

[0538] A mixture of 2 g of mesylate and 5 ml of DMSO in a flask washeated at 80° C. for 22.4 hours. HPLC analysis of the reaction mixtureindicated no starting material was detected. To the reaction was addedwater (10 ml) and the precipitate was extracted with methylene chloridethree times. The combined methylene chloride layers were washed withwater, dried over magnesium sulfate, and concentrated to give theenester.

EXAMPLE 42

[0539] In a 50 mL pear-shaped flask under stirring the enester ofFormula IIA (1.07 g assaying 74.4% enester), trichloroacetamide (0.32g), dipotassium hydrogen phosphate (0.70 g) as solid were mixed withmethylene chloride (15.0 mL). A clear solution was obtained. Hydrogenperoxide (30% by weight; 5.0 mL) was added via a pipet over a 1 min.period. The resulting mixture was stirred for 6 hrs. at room temperatureat which point HPLC analysis showed that the ratio of epoxymexrenone toenester in the reaction mixture was approximately 1:1. Additionaltrichloroacetamide (0.32 g) was added to the reaction mixture andreaction continued under agitation for 8 more hours after which time theremaining proportion of enester was shown to have been reduced to 10%.Additional trichloroacetamide (0.08 g) was added and the reactionmixture was allowed to stand overnight at which point only 5% ofunreacted enester remained relative to epaxymexrenone in the mixture.

EXAMPLE 43

[0540] Enester of Formula IIA (5.4 g, assaying 74.4% enester) was addedto a 100 mL reactor. Trichloroacetamide (4.9 g) and dipotassium hydrogenphosphate (3.5 g) both in solid form were added to the enester followedby methylene chloride (50 mL). The mixture was cooled to 15° C. and a30% hydrogen peroxide (25 g) was added over a ten min. period. Thereaction mixture was allowed to come to 20° C. and stirred at thattemperature for 6 hrs., at which point conversion was checked by HPLC.Remaining enester was determined to be less than 1% by weight.

[0541] The reaction mixture was added to water (100 mL), the phases wereallowed to separate, and the methylene chloride layer was removed.Sodium hydroxide (0.5 N; 50 mL) was added to the methylene chloridelayer. After 20 min. the phases were allowed to separate HCl (0.5 N; 50mL) was added to the methylene chloride layer after which the phaseswere allowed to separate and the organic phase was washed with saturatedbrine (50 mL). The methylene chloride layer was dried over anhydrousmagnesium sulfate and the solvent removed. A white solid (5.7 g) wasobtained. The aqueous sodium hydroxide layer was acidified and extractedand the extract worked up to yield an additional 0.2 g of product. Yieldof epoxymexrenone was 90.2%.

EXAMPLE 44

[0542] Enester of Formula IIA was converted to epoxymexrenone in themanner described in Example 43 with the following differences: theinitial charge comprised of enester (5.4 g assaying 74.4% enester),trichloroacetamide (3.3 g), and dipotassium hydrogen phosphate (3.5 g).Hydrogen peroxide solution (12.5 mL) was added. The reaction wasconducted overnight at 20° C. after which HPLC showed a 90% conversionof enester to epoxymexrenone. Additional trichloroacetamide (3.3 g) and30% hydrogen peroxide (5.0 mL) was added and the reaction carried outfor an additional 6 hrs. at which point the residual enester was only 2%based on the enester charge. After work up as described in Example 43,5.71 g of epoxymexrenone resulted.

EXAMPLE 45

[0543] The enester of Formula IIA was converted to epoxymexrenone in themanner generally described in Example 43. In the reaction of thisExample, enester charge was 5.4 g (assaying 74.4% enester), thetrichloroacetamide charge was 4.9 g, hydrogen peroxide charge was 25 g,dipotassium hydrogen phosphate charge was 3.5 g. The reaction was run at20° C. for 18 hrs. The residual enester was less than 2%. After work up,5.71 g of epoxymexrenone resulted.

EXAMPLE 46

[0544] Enester of Formula IIA was converted to epoxymexrenone in themanner described in Example 43 except that the reaction temperature inthis Example was 28° C. The materials charged in the reactor includedenester (2.7 g), trichloroacetamide (2.5 g), dipotassium hydrogenphosphate (1.7 g), hydrogen peroxide (17.0 g) and methylene chloride (50mL). After 4 hrs. reaction, unreacted enester was only 2% based on theenester charge. After work up as described in Example 43, 3.0 g ofepoxymexrenone was obtained.

EXAMPLE 47

[0545] Enester of Formula IIA (17 g assaying 72% enester) was dissolvedin methylene chloride (150 mL) after which trichloroacetamide (14.9 g)was added under slow agitation. The temperature of the mixture wasadjusted to 25° C. and the solution of dipotassium hydrogen phosphate(10.6 g) in water (10.6 mL) was stirred into the enester substratesolution under 400 rpm agitation. Hydrogen peroxide (30% by weightsolution; 69.4 mL) was added to thesubstrate/phosphate/trichloroacetamide solution over a 3-5 min. period.No exotherm or oxygen evolution was observed. The reaction mixture thusprepared was stirred at 400 rpm and 25° C. for 18.5 hrs. No oxygenevolution was observed throughout the course of the reaction. Thereaction mixture was diluted with water (69.4 mL) and the mixturestirred at about 250 rpm for 15 min. No temperature control wasnecessary for this operation and it was conducted essentially at roomtemperature (any temperature in the range of 5-25° C. being acceptable).The aqueous and organic layers were allowed to separate and the lowermethylene chloride layer was removed.

[0546] The aqueous layer was back extracted with methylene chloride(69.4 mL) for 15 min. under agitation of 250 rpm. The layers wereallowed to separate and the lower methylene chloride layer was removed.The aqueous layer (177 g; pH=7) was submitted for hydrogen peroxidedetermination. The result (12.2%) indicating that only 0.0434 mol ofhydrogen peroxide were consumed in the reaction was 0.0307 mol ofolefin. Back extraction with a small amount of methylene chloride volumewas sufficient to insure no loss of epoxymexrenone in the aqueous layer.This result was confirmed with the application of a second largemethylene chloride extraction in which only trichloroacetamide wasrecovered.

[0547] The combined methylene chloride solutions from the abovedescribed extractions were combined and washed with 3% by weight sodiumsulfite solution (122 mL) for at least 15 min. at about 250 rpm. Anegative starch iodide test (KI paper; no color observed; in a positivetest a purple coloration indicates the presence of peroxide) wasobserved at the end of the stir period.

[0548] The aqueous and organic layers were allowed to separate and thelower methylene chloride layer removed. The aqueous layer (pH=6) wasdiscarded. Note that addition of sodium sulfite solution can cause aslight exotherm so that such addition should be carried out undertemperature control.

[0549] The methylene chloride phase was washed with 0.5 N sodiumhydroxide (61 mL) for 45 min. at about 250 rpm and a temperature in therange of 15-25° C. (pH=12-13). Impurities derived fromtrichloroacetamide were removed in this process. Acidification of thealkaline aqueous fraction followed by extraction of the methylenechloride confirmed that very little epoxymexrenone was lost in thisoperation.

[0550] The methylene chloride phase was washed once with 0.1 Nhydrochloric acid (61 mL) for 15 min. under 250 rpm agitation at atemperature in the range 15-25° C. The layers were then allowed toseparate and the lower methylene chloride layer removed and washed againwith 10% by weight aqueous sodium chloride (61 mL) for 15 min at 250 rpmat a temperature in the range of 15-25° C. Again the layers were allowedto separate and the organic layer removed. The organic layer wasfiltered through a pad of Solkafloc and then evaporated to dryness underreduced pressure. Drying was completed with a water bath temperature of65° C. An off-white solid (17.95 g) was obtained and submitted for HPLCassay. Epoxymexrenone assay was 66.05%. An adjusted molar yield for thereaction was 93.1%.

[0551] The product was dissolved in hot methyl ethyl ketone (189 mL) andthe resulting solution was distilled at atmospheric pressure until 95 mLof the ketone solvent had been removed. The temperature was lowered to50° C. as the product crystallized. Stirring was continued at 50° C. for1 hr. The temperature was then lowered to 20-25° C. and stirringcontinued for another 2 hrs. The solid was filtered and rinsed with MEK(24 mL) and the solid dried to a constant weight of 9.98 g, which byHPLC assay contain 93.63% epoxymexrenone. This product was re-dissolvedin hot MEK (106 mL) and the hot solution filtered through a 10 micronline filter under pressure. Another 18 mL of MEK was applied as a rinseand the filtered MEK solution distilled at atmospheric pressure until 53mL of solvent had been removed. The temperature was lowered to 50° C. asthe product crystallized; and stirring was continued at 50° C. for 1 hr.The temperature was then lowered to 20-25° C. and held at thattemperature while stirring was continued for another 2 hrs. The solidproduct was filtered and rinsed with MEK (18 mL). The solid product wasdried to a constant weight of 8.32 g which contained 99.6%epoxymexrenone per quantitative HPLC assay. The final loss on drying wasless than 1.0%. Overall yield of epoxymexrenone in accordance with thereaction and work up of this Example is 65.8%. This overall yieldreflected a reaction yield of 93%, an initial crystallization recoveryof 78.9%, and a recrystallization recovery of 89.5%.

EXAMPLE 48 Epoxidation of Formula IIA Using Toluene

[0552] The enester of Formula IIA was converted to eplerenone in themethod generally described in Example 46 except that toulene was used asthe solvent. The materials charged to the reactor included enester (2.7g) trichloroacetamide (2.5 g), dipotassium hydrogen phosphate (1.7 g),hydrogen peroxide (17.0 g) and toulene (50 ml). The reaction was allowedto exotherm to 28° C. and was complete in 4 hours. The resulting threephase mixture was cooled to 15° C., filtered, washed with water anddried in vacuo to yield 2.5 g of product.

EXAMPLE 49 Epoxidation of 9,11-Dienone

[0553] A compound designated XVIIA (compound XVII wherein —A—A— and—B—B— are both —CH₂—CH₂—) (40.67 g) was dissolved in methylene chloride(250 mL) in a one liter 3 necked flask and cooled by ice salt mixtureexternally. Dipotassium phosphate (22.5 g), and trichloroacetonitrile(83.5 g) were added and mixture cooled to 2° C. after which 30% Hydrogenperoxide (200 g) was slowly added over a period of 1 hour. The reactionmixture was stirred at 12° for 8 hours and 14 hours at room temperature.A drop of the organic layer was taken and checked for any starting enoneand was found to be <0.5%. Water (400 mL) was added, stirred for 15 min.and layers separated. The organic layer was washed successively with 200mL of potassium iodide (10%), 200 mL of sodium thiosulfate (10%) and 100mL of saturated sodium bicarbonate solution separating layers each time.The organic layer was dried over anhydrous magnesium sulfate andconcentrated to yield crude epoxide (41 g). The product crystallizedfrom ethyl acetate:methylene chloride to give 14.9 g of pure material.

EXAMPLE 50 Epoxidation of Compound XVIIA Using m-chloroperbenzoic Acid

[0554] Compound XVIIA (18.0 g) was dissolved in 250 mL of methylenechloride and cooled to 10° C. Under stirring solid m-chloroperbenzoicacid, (50-60% pure, 21.86 g) was added during 15 min. No rise intemperature was observed. The reaction mixture was stirred for 3 hoursand checked for the presence of the dienone. The reaction mixture wastreated successively with sodium sulfite solution (10%), sodiumhydroxide solution (0.5N), hydrochloric acid solution (5%) and finallywith 50 mL of saturated brine solution. After drying with anhydrousmagnesium sulfate and evaporation, 17.64 g of the epoxide resulted andwas used directly in the next step. The product was found to containBaeyer-Villiger oxidation product that had to be removed by triturationfrom ethyl acetate followed by crystallization from methylene chloride.On a 500 g scale, the precipitated m-chlorobenzoic acid was filteredfollowed by the usual work up.

EXAMPLE 51 Epoxidation of Compound XVIIA Using Trichloroacetamide

[0555] Compound XVIIA (2 g) was dissolved in 25 mL of methylenechloride. Trichloroacetamide (2 g), dipotassium phosphate (2 g) wereadded. Under stirring at room temperature 30% hydrogen peroxide (10 mL)was added and stirring continued for 18 hours to yield the epoxide (1.63g). Baeyer-Villiger product was not formed.

EXAMPLE 52

[0556] Potassium hydroxide (56.39 g; 1005.03 mmol; 3.00 eq.) was chargedto a 2000 mL flask and slurried with dimethylsulfoxide (750.0 mL) atambient temperature. A trienone corresponding to Formula XX (wherein R³is H and —A—A— and —B—B— are each —CH₂—CH₂—) (100.00 g; 335.01 mmol;1.00 eq.) was charged to the flask together with THF (956.0 mL).Trimethylsulfonium methylsulfate (126.14 g; 670.02 mmol; 2.00 eq.) wascharged to the flask and the resulting mixture heated at reflux, 80 to85° C. for 1 hr. Conversion to the 17-spirooxymethylene was checked byHPLC. THF approximately 1 L was stripped from the reaction mixture undervacuum after which water (460 mL) was charged over a 30 min. periodwhile the reaction mixture was cooled to 15° C. The resulting mixturewas filtered and the solid oxirane product washed twice with 200 mLaliquots of water. The product was observed to be highly crystalline andfiltration was readily carried out. The product was thereafter driedunder vacuum at 40° C. 104.6 g of the 3-methyl enol etherΔ-5,6,9,11,-17-oxirane steroid product was isolated.

EXAMPLE 53

[0557] Sodium ethoxide (41.94 g; 616.25 mmol; 1.90 eq.) was charged to adry 500 mL reactor under a nitrogen blanket. Ethanol (270.9 mL) wascharged to the reactor and the sodium methoxide slurried in the ethanol.Diethyl malonate (103.90 g; 648.68 mmol; 2.00 eq.) was charged to theslurry after which the oxirane steroid prepared in the manner describedin Example 52 (104.60 g; 324.34 mmol; 1.00 eq.) was added and theresulting mixture heated to reflux, i.e., 80 to 85° C. Heating wascontinued for 4 hrs. after which completion of the reaction was checkedby HPLC. Water (337.86 mL) was charged to the reaction mixture over a 30min. period while the mixture was being cooled to 15° C. Stirring wascontinued for 30 min. and then the reaction slurry filtered producing afilter cake comprising a fine amorphous powder. The filter cake waswashed twice with water (200 mL each) and thereafter dried at ambienttemperature under vacuum. 133.8 g of the 3-methylenolether-Δ5,6,9,11,-17-spirolactone-21-methoxycarbonyl intermediate wasisolated.

EXAMPLE 54

[0558] The 3-methylenolether-Δ5,6,9,11,-17-spirolactone-21-methoxycarbonyl intermediate(Formula XVIII where R³ is H and —A—A— and —B—B— are each —CH₂—CH₂—;133.80 g; 313.68 mmol; 1.00 eq., as produced in Example 53, was chargedto the reactor together with sodium chloride (27.50 g; 470.52 mmol; 1.50eq.) dimethyl formamide (709 mL) and water (5 mL) were charged to a 2000mL reactor under agitation. The resulting mixture was heated to reflux,138 to 142° C. for 3 hrs. after which the reaction mixture was checkedfor completion of the reaction by HPLC. Water was thereafter added tothe mixture over a 30 min. period while the mixture was being cooled to15° C. Agitation was continued for 30 min. after which the reactionslurry was filtered recovering amorphous solid reaction product as afilter cake. The filter cake was washed twice (200 mL aliquots of water)after which it was dried. The product 3-methylenolether-17-spirolactonewas dried yielding 91.6 g (82.3% yield; 96 area % assay).

EXAMPLE 55

[0559] The enol ether produced in accordance with Example 54 (91.60 g;258.36 mmol; 1.00 eq.) ethanol (250 ML) acetic acid (250 mL) and water(250 mL) were charged to a 2000 mL reactor and the resulting slurryheated to reflux for 2 hrs. Water (600 mL) was charged over a 30 min.period while the reaction mixture was being cooled to 15° C. Thereaction slurry was thereafter filtered and the filter cake washed twicewith water (200 mL aliquots). The filter cake was then dried; 84.4 g ofproduct 3-keto Δ4,5,9,11,-17-spirolactone was isolated (compound ofFormula XVII where R³ is H and —A—A— and —B—B— are —CH₂—CH₂—; 95.9%yield).

EXAMPLE 56

[0560] Compound XVIIA (1 kg; 2.81 moles) was charged together withcarbon tetrachloride (3.2 L) to a 22 L 4-neck flask. N-bromo-succinamide(538 g) was added to the mixture followed by acetonitrile (3.2 L). Theresulting mixture was heated to reflux and maintained at the 68° C.reflux temperature for approximately 3 hrs. producing a clear orangesolution. After 5 hrs. of heating, the solution turned dark. After 6hrs. the heat was removed and the reaction mixture was sampled. Thesolvent was stripped under vacuum and ethyl acetate (6 L) added to theresidue in the bottom of the still. The resultant mixture was stirredafter which a 5% sodium bicarbonate solution (4 L) was added and themixture stirred for 15 min. after which the phases were allowed tosettle. The aqueous layer was removed and saturated brine solution (4 L)introduced into the mixture which was then stirred for 15 min. Thephases were again separated and the organic layer stripped under vacuumproducing a thick slurry. Dimethylformamide (4 L) was then added andstripping continued to a pot temperature of 55° C. The still bottomswere allowed to stand overnight and DABCO (330 g) and lithium bromide(243 g) added. The mixture was then heated to 70° C. After one andone-half hrs. heating, a liquid chromatography sample was taken andafter 3.50 hrs. heating, additional DABCO (40 g) was added. After 4.5hrs. heating, water (4 L) was introduced and the resulting mixture wascooled to 15° C. The slurry was filtered and the cake washed with water(3 L) and dried on the filter overnight. The wet cake (978 g) wascharged back into the 22 L flask and dimethylformamide (7 L) added. Themixture thus produced was heated to 105° C. at which point the cake hadbeen entirely taken up into solution. The heat was removed and themixture in the flask was stirred and cooled. Ice water was applied tothe reactor jacket and the mixture within the reactor cooled to 14° C.and held for two hours. The resulting slurry was filtered and washedtwice with 2.5 L aliquots of water. The filter cake was dried undervacuum overnight. A light brown solid product 510 g was obtained.

EXAMPLE 57

[0561] To a 2 L 4-neck flask were charged: 9,11-epoxy canrenone asproduced in Example 49, 50, or 51 (100.00 g; 282.1 mmol; 1.00 eq.),dimethylformamide (650.0 mL), lithium chloride (30.00 g; 707.7 mmol;2.51 eq.), and acetone cyanohydrin (72.04 g; 77.3 mL; 846.4 mmol; 3.00eq.). The resulting suspension was mechanically stirred and treated withtetramethyl guanidine (45.49 g; 49.6 mL; 395.0 mmol; 1.40 eq.). Thesystem was then filtered with a water cooled condenser and a dry icecondenser (filled with dry ice in acetone) to prevent escape of HCN. Thevent line from the dry ice condenser passed into a scrubber filled witha large excess of chlorine bleach. The mixture was heated to 80° C.

[0562] After 18 hrs., a dark reddish-brown solution was obtained whichwas cooled to room temperature with stirring. During the coolingprocess, nitrogen was sparged into the solution to remove residual HCNwith the vent line being passed into bleach in the scrubber. After twohrs. the solution was treated with acetic acid (72 g) and stirred for 30min. The crude mixture was then poured into ice water (2 L) withstirring. The stirred suspension was further treated with 10% aqueousHCl (400 mL) and stirred for 1 hr. Then the mixture was filtered to givea dark brick-red solid (73 g). The filtrate was placed in a 4 Lseparatory funnel and extracted with methylene chloride (3×800 mL); andthe organic layers were combined and back extracted with water (2×2 L).The methylene chloride solution was concentrated in vacuo to give 61 gof a dark red oil.

[0563] After the aqueous wash fractions were allowed to sit overnight, aconsiderable precipitate developed. This precipitate was collected byfiltration and determined to be pure product enamine (14.8 g).

[0564] After drying the original red solid (73 g) was analyzed by HPLCand it was determined that the major component was the9,11-epoxyenamine. HPLC further showed that enamine was the majorcomponent of the red oil obtained from methylene chloride workup.Calculated molar yield of enamine was 46%.

EXAMPLE 58

[0565] 9,11-epoxyenamine (4.600 g; 0.011261 mol; 1.00 eq.) as preparedin accordance with Example 57 was introduced into a 1000 mL round bottomflask. Methanol (300 mL) and 0.5% by weight aqueous HCl (192 mL) wereadded to the mixture which was thereafter refluxed for 17 hrs. Methanolwas thereafter removed under vacuum reducing the amount of material inthe still pot to 50 mL and causing a white precipitate to be formed.Water (100 mL) was added to the slurry which was thereafter filteredproducing a white solid cake which was washed three times with water.Yield of solid 9,11-epoxydiketone product was 3.747 g (81.3%).

EXAMPLE 59

[0566] The epoxydiketone prepared in accordance with Example 58 (200 mg;0.49 mmol) was suspended in methanol (3 mL) and1,8-diazabicyclo[5.4.0]undec-7-ene(DBU) added to the mixture. Uponheating under reflux for 24 hrs. the mixture became homogeneous. It wasthen concentrated to dryness at 30° C. on a rotary evaporator and theresidue partitioned between methylene chloride and 3.0 N HCl.Concentration of the organic phase yielded a yellow solid (193 mg) whichwas determined to be 22% by weight epoxy mexrenone. The yield was 20%.

EXAMPLE 60

[0567] To 100 mg of the diketone suspended in 1.5 mL of methanol wasadded 10 microliters (0.18 eq) of a 25% (w/w) solution of sodiummethoxide in methanol. The solution was heated to reflux. After 30 min.no diketone remained and the 5-cyanoester was present. To the mixturewas added 46 microliters of 25% (w/w) sodium methanol solution inmethanol. The mixture was heated at reflux for 23 hours at which timethe major product was eplerenone as judged by HPLC.

EXAMPLE 61

[0568] To 2 g of the diketone suspended in 30 ml of dry methanol wasadded 0.34 mL of triethylamine. The suspension was heated at reflux for4.5 hours. The mixture was stirred at 25° C. for 16 hours. The resultingsuspension was filtered to give 1.3 g of the 5-cyanoester as a whitesolid.

[0569] To 6.6 g of the diketone suspended in 80 mL of methanol was added2.8 mL of triethylamine. The mixture was heated at reflux for 4 hoursand was stirred at 25× for 88 hours during which time the productcrystallized from solution. Filtration followed by a methanol wash gave5.8 g of the cyanoester as a white powder. The material wasrecrystallized from chloroform/methanol to give 3.1 g of crystallinematerial which was homogeneous by HPLC.

[0570] In view of the above, it will be seen that the several objects ofthe invention are achieved and other advantageous results attained.

[0571] As various changes could be made in the above compositions andprocesses without departing from the scope of the invention, it isintended that all matter contained in the above description and shown inthe accompanying drawings shall be interpreted as illustrative and notin a limiting sense.

What is claimed is:
 1. A process for the preparation of a compound ofFormula II:

wherein —A—A— represents the group —CHR⁴—CHR⁵— or —CR⁴—CR⁵— R³, R⁴ andR⁵ are independently selected from the group consisting of hydrogen,halo, hydroxy, lower alkyl, lower alkoxy, hydroxyalkyl, alkoxyalkyl,hydroxy carbonyl, cyano, aryloxy, R¹ represents an alpha-oriented loweralkoxycarbonyl or hydroxycarbonyl radical, —B—B— represents the group—CHR⁶—CHR⁷— or an alpha- or beta-oriented group:

 where R⁶ and R⁷ are independently selected from the group consisting ofhydrogen, halo, lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl,hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl, cyano, aryloxy,and R⁸ and R⁹ are independently selected from the group consisting ofhydrogen, halo, lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl,hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl, cyano, aryloxy, orR⁸ and R⁹ together comprise a carbocyclic or heterocyclic ringstructure, or R⁸ or R⁹ together with R⁶ or R⁷ comprise a carbocyclic orheterocyclic ring structure fused to the pentacyclic D ring. the processcomprising: removing an 11α-leaving group from a compound of Formula IV:

wherein —A—A—, R¹, R³, —B—B—, R⁸, and R⁹ are as defined above, and R² isa leaving group the abstraction of which is effective for generating adouble bond between the 9- and 11-carbon atoms.
 2. A process as setforth in claim 1 wherein said compound of Formula II corresponds toFormula IIA:

wherein: —A—A— represents the group —CH₂—CH₂— or —CH═CH—, —B—B—represents the group —CH₂—CH₂— or an alpha- or beta-oriented group ofFormula IIIA:

R¹ represents an alpha-oriented lower alkoxycarbonyl radical, Xrepresents two hydrogen atoms or oxo, Y¹ and Y² together represent theoxygen bridge —O—, or Y¹ represents hydroxy, and Y² represents hydroxy,lower alkoxy or, if X represents H_(2,) also lower alkanoyloxy, andsalts of compounds in which X represents oxo and Y² represents hydroxy-,the process comprising: contacting a solution comprising a loweralkanoic acid and a salt of a lower alkanoic acid with a compoundcorresponding to Formula IVA:

wherein —A—A—, R¹, —B—B—, X, Y¹ and Y² are as defined in Formula IIA andR² is lower alkylsulfonyloxy or acyloxy.
 3. A process as set forth inclaim 1 wherein said compound of Formula IV is Methyl Hydrogen17α-Hydroxy-11α-(methylsulfonyl)oxy-3-oxopregn-4-ene-7α,21-dicarboxylate,γ-Lactone and said compound of Formula II is Methyl Hydrogen17α-Hydroxy-3-oxopregna-4,9(11)-diene-7α,21-dicarboxylate, γ-Lactone. 4.A process for the preparation of a compound of Formula IV:

wherein —A—A— represents the group —CHR⁴—CHR⁵— or —CR⁴═CR⁵— R³, R⁴ andR⁵ are independently selected from the group consisting of hydrogen,halo, hydroxy, lower alkyl, lower alkoxy, hydroxyalkyl, alkoxyalkyl,hydroxy carbonyl, cyano, aryloxy, R¹ represents an alpha-oriented loweralkoxycarbonyl or hydroxycarbonyl radical, —B—B— represents the group—CHR⁶—CHR⁷— or an alpha- or beta-oriented group:

 where R⁶ and R⁷ are independently selected from the group consisting ofhydrogen, halo, lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl,hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl, cyano, aryloxy,and R⁸ and R⁹ are independently selected from the group consisting ofhydrogen, halo, lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl,hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl, cyano, aryloxy orR⁸ and R⁹ together comprise a carbocyclic or heterocyclic ringstructure, or R⁸ or R⁹ together with R⁶ or R⁷ comprise a carbocyclic orheterocyclic ring structure fused to the pentacyclic D ring, and R² islower alkylsulfonyloxy or acyloxy or a halide. the process comprising:reacting a lower alkylsulfonylating or acylating reagent or a halidegenerating agent such as thionyl halide, sulfuryl halide, or oxalylhalide with a compound of Formula V

wherein —A—A—, R¹, R³, —B—B—, R⁸, and R⁹ are as defined above.
 5. Aprocess as set forth in claim 4 wherein said compound of Formula IVcorresponds to Formula IVA:

wherein: —A—A— represents the group —CH₂—CH₂— or —CH═CH—, R¹ representsan alpha-oriented lower alkoxycarbonyl radical, R² represents loweralkylsulfonyloxy or acyloxy, —B—B— represents the group —CH₂—CH₂— or analpha- or beta-oriented group:

X represents two hydrogen atoms or oxo, Y¹ and Y² together represent theoxygen bridge —O—, or Y¹ represents hydroxy, and Y² represents hydroxy,lower alkoxy or, if X represents H₂, also lower alkanoyloxy, and saltsof compounds in which X represents oxo and Y² represents hydroxy-, theprocess comprising: reacting a lower alkylsulfonyl or acyl halide in thepresence of a hydrogen halide scavenger with a compound corresponding tothe formula:

wherein —A—A—, R¹, —B—B—, X, Y¹, and Y² are as defined in Formula IVA.6. A process as set forth in claim 4 wherein said compound of Formula IVis Methyl Hydrogen17α-Hydroxy-11α-(methylsulfonyl)oxy-3-oxopregn-4-ene-7α,21-dicarboxylate,γ-Lactone and said compound of Formula V is Methyl Hydrogen 11α,17α-Dihydroxy-3-oxopregn-4-ene-7α,21-dicarboxylate, γ-Lactone.
 7. Aprocess for the preparation of a compound of Formula V:

wherein —A—A— represents the group —CHR⁴—CHR⁵— or —CR⁴═CR⁵— R³, R⁴ andR⁵ are independently selected from the group consisting of hydrogen,halo, hydroxy, lower alkyl, lower alkoxy, hydroxyalkyl, alkoxyalkyl,hydroxycarbonyl, cyano, aryloxy, R¹ represents an alpha-oriented loweralkoxycarbonyl or hydroxycarbonyl radical, —B—B— represents the group—CHR⁶—CHR⁷— or an alpha- or beta-oriented group:

 where R⁶ and R⁷ are independently selected from the group consisting ofhydrogen, halo, lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl,hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl, cyano, aryloxy,and R⁸ and R⁹ are independently selected from the group consisting ofhydrogen, halo, lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl,hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl, cyano, aryloxy, orR⁸ and R⁹ together comprise a carbocyclic or heterocyclic ringstructure, or R⁸ or R⁹ together with R⁶ or R⁷ comprise a carbocyclic orheterocyclic ring structure fused to the pentacyclic D ring. the processcomprising: reacting a compound of Formula VI with an alkali metalalkoxide corresponding to the formula R¹⁰OM wherein M is alkali metaland R¹⁰O— corresponds to the alkoxy substituent of R¹, said compound ofFormula VI having the structure:

wherein —A—A—, R³, —B—B—, R³, and R⁹ are as defined above.
 8. A processas set forth in claim 7 wherein the compound of Formula V corresponds tothe formula:

wherein —A—A— represents the group —CH₂—CH₂— or —CH═CH—, R¹ representsan alpha-oriented lower alkoxycarbonyl radical, —B—B— represents thegroup —CH₂—CH₂— or an alpha- or beta-oriented group:

X represents two hydrogen atoms or oxo, Y¹and Y² together represent theoxygen bridge —O—, or y¹ represents hydroxy, and Y² represents hydroxy,lower alkoxy or, if X represents H₂, also lower alkanoyloxy, and saltsof compounds in which X represents oxo and Y² represents hydroxy-, theprocess comprising: reacting a compound of Formula VIA with an alkalimetal alkoxide corresponding to the formula R¹⁰OM in the presence of analcohol having the formula R¹⁰OH, wherein M is alkali metal and R¹⁰O—corresponds to the alkoxy substituent of R¹, said compound of Formula VIhaving the structure:

wherein —A—A—, —B—B—, Y¹, Y² and X are as defined in Formula VA.
 9. Aprocess as set forth in claim 7 wherein the compound of Formula V isMethyl Hydrogen 11α,17α-Dihydroxy-3-oxopregn-4-ene-7α,21-dicarboxylate,γ-Lactone and the compound of Formula VI is4′S(4′α),7′α-Hexadecahydro-11′α-hydroxy-10′β,13′β-dimethyl-3′,5,20′-trioxospiro[furan-2(3H),17′β-[4,7]methano[17H]cyclopenta[a]phenanthrene]-5′β(2′H)-carbonitrile.10. A process as set forth in claim 7 wherein cyanide ion is formed as aby-product of the reaction, the process further comprising removal ofcyanide ion from the reaction zone during the reaction to reduce theextent of any reaction of cyanide ion with the product of Formula V. 11.A process as set forth in claim 10 wherein cyanide ion is removed fromthe reaction by precipitation with a precipitating agent.
 12. A processas set forth in claim 11 wherein said reaction is carried out in asolvent medium, and said precipitating agent comprises a salt comprisinga cation which forms a cyanide compound of lower solubility in saidmedium than the solubility of the precipitating agent therein.
 13. Aprocess as set forth in claim 12 wherein said cation is selected fromthe group consisting of alkaline earth metal ions and transition metalions.
 14. A process for the preparation of a compound of Formula VI:

wherein —A—A— represents the group —CHR⁴—CHR⁵— or —CR⁴═CR⁵— R³, R⁴ andR⁵ are independently selected from the group consisting of hydrogen,halo, hydroxy, lower alkyl, lower alkoxy, hydroxyalkyl, alkoxyalkyl,hydroxycarbonyl, cyano, aryloxy, —B—B— represents the group —CHR⁶—CHR⁷—or an alpha- or beta-oriented group:

 where R⁶ and R⁷ are independently selected from the group consisting ofhydrogen, halo, lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl,hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl, cyano, aryloxy,and R⁸ and R⁹ are independently selected from the group consisting ofhydrogen, halo, lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl,hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl, cyano, aryloxy, orR⁸ and R⁹ together comprise a carbocyclic or heterocyclic ringstructure, or R⁸or R⁹ together with R⁶ or R⁷ comprise a carbocyclic orheterocyclic ring structure fused to the pentacyclic D ring. the processcomprising: hydrolyzing a compound corresponding to Formula VII:

wherein —A—A—, R³, —B—B—, R⁸, and R⁹ are as defined above.
 15. A processas set forth in claim 14 wherein said compound of Formula VI correspondsto the formula:

wherein: —A—A— represents the group —CH₂—CH₂— or —CH═CH—, —B—B—represents the group —CH₂—CH₂— or an alpha- or beta-oriented group:

X represents two hydrogen atoms or oxo, Y¹ and Y²together represent theoxygen bridge —C—, or Y¹ represents hydroxy, and Y² represents hydroxy,lower alkoxy or, if X represents H₂, also lower alkanoyloxy, and saltsof compounds in which X represents oxo and Y² represents hydroxy-, theprocess comprising: hydrolyzing a compound of Formula VIIA in thepresence of an acid and an organic solvent and/or water, said compoundof Formula VIIA having the structure:

wherein —A—A—, —B—B—, Y¹, Y², and X are as defined in Formula VIA.
 16. Aprocess as set forth in claim 14 wherein said compound of Formula VI is4′S(4′α),7′α-Hexadecahydro-11′α-hydroxy-10′β,13′β-dimethyl-3′,5,20′-trioxospiro[furan-2(3H),17′β-[4,7]methano[17H]cyclopenta[a]phenanthrene]-5′β(2′H)-carbonitrileand said compound of Formula VII is5′R(5′α),7′β-20′-Aminohexadecahydro-11′-hydroxy-10′α,13′α-dimethyl-3′,5-dioxospiro[furan-2(3H),17′α(5′H)-[7,4]metheno[4H]cyclopenta[a]phenanthrene]-5′-carbonitrile.17. A process for the preparation of a compound of Formula VII:

wherein —A—A— represents the group —CHR⁴—CHR⁵— or —CR⁴═CR′— R^(3,) R⁴and R⁵ are independently selected from the group consisting of hydrogen,halo, hydroxy, lower alkyl, lower alkoxy, hydroxyalkyl, alkoxyalkyl,hydroxycarbonyl, cyano, aryloxy, —B—B— represents the group —CHR⁶—CHR⁷or an alpha- or beta-oriented group:

 where R⁶ and R⁷ are independently selected from the group consisting ofhydrogen, halo, lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl,hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl, cyano, aryloxy,and R⁸ and R⁹ are independently selected from the group consisting ofhydrogen, halo, lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl,hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl, cyano, aryloxy, orR⁸ and R⁹ together comprise a carbocyclic or heterocyclic ringstructure, or R⁸ or R⁹ together with R⁶ or R⁷ comprise a carbocyclic orheterocyclic ring structure fused to the pentacyclic D ring. the processcomprising: reacting a compound of Formula VIII with a source of cyanideion in the presence of an alkali metal salt, said compound of FormulaVIII having the structure:

wherein —A—A—, R³, —B—B—, R⁸, and R⁹ are as defined above.
 18. A processas set forth in claim 17 wherein said compound of Formula VIIcorresponds to Formula VIIA:

wherein: —A—A— represents the group —CH₂—CH₂— or —CH═CH—, —B—B—represents the group —CH₂—CH₂— or an alpha- or beta-oriented group:

X represents two hydrogen atoms or oxo, Y¹ and Y² together represent theoxygen bridge —O—, or Y¹ represents hydroxy, and Y² represents hydroxy,lower alkoxy or, if X represents H₂, also lower alkanoyloxy, and saltsof compounds in which X represents oxo and Y² represents hydroxy-, theprocess comprising: reacting a cyanide source such as ketone cyanohydrinin the presence of LiCl in the presence of a base with an 11α-hydroxycompound corresponding to the formula:

wherein —A—A—, —B—B—, Y¹, Y², and X are as defined in Formula VIIA. 19.A process as set forth in claim 17 wherein said compound of Formula VIIis5′R(5′α),7′β-20′-Aminohexadecahydro-11′β-hydroxy-10′α,13′α-dimethyl-3′,5-dioxospiro[furan-2(3H),17′α(5′H)-[7,4]metheno[4H]cyclopenta[a]phenanthrene]-5′-carbonitrileand said compound of Formula VIII is11α,17α-Dihydroxy-3-oxopregna-4,6-diene-21-carboxylic Acid, γ-Lactone.20. A process as set forth in claim 17 wherein said source of cyanideion comprises an alkali metal cyanide, the reaction between saidcompound of Formula VIII and cyanide ion being carried out in thepresence of an acid and water.
 21. A process for the preparation of acompound of Formula VIII

wherein —A—A— represents the group —CHR⁴—CHR⁵— or —CR⁴═CR⁵— R³, R⁴ andR⁵ are independently selected from the group consisting of hydrogen,halo, hydroxy, lower alkyl, lower alkoxy, hydroxyalkyl, alkoxyalkyl,hydroxycarbonyl, cyano, aryloxy, —B—B— represents the group —CHR⁶—CHR⁷—or an alpha- or beta-oriented group:

 where R⁶ and R⁷ are independently selected from the group consisting ofhydrogen, halo, lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl,hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl, cyano, aryloxy,and R⁸ and R⁹ are independently selected from the group consisting ofhydrogen, halo, lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl,hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl, cyano, aryloxy, orR⁸ and R⁹ together comprise a carbocyclic or heterocyclic ringstructure, or R⁸ and R⁹ together with R⁶ or R⁷ comprise a carbocyclic orheterocyclic ring structure fused to the pentacyclic D ring, the processcomprising: oxidizing a substrate compound corresponding to Formula X byfermentation in the presence of a microorganism effective forintroducing an 11-hydroxy group into said substrate in α-orientation,said substrate corresponding to the formula:

wherein —A—A—, R¹, R³, —B—B—, R⁸, and R⁹ are as defined above.
 22. Aprocess as set forth in claim 21 wherein said compound of Formula VIIIis 11α,17α-Dihydroxy-3-oxopregna-4,6-diene-21-carboxylic Acid,γ-Lactone.
 23. A process for the preparation of a mexrenone derivativecorresponding to the formula:

wherein —A—A— represents the group —CHR⁴—CHR⁵— or —CR⁴═CR⁵— R³, R⁴ andR⁵ are independently selected from the group consisting of hydrogen,halo, hydroxy, lower alkyl, lower alkoxy, hydroxyalkyl, alkoxyalkyl,hydroxycarbonyl, cyano, aryloxy, R¹ represents an alpha-oriented loweralkoxycarbonyl or hydroxycarbonyl radical, —B—B— represents the group—CHR⁶—CHR⁷— or an alpha- or beta-oriented group:

 where R⁶ and R⁷ are independently selected from the group consisting ofhydrogen, halo, lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl,hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl, cyano, aryloxy,the process comprising: reacting a compound of Formula XIV with analkali metal alkoxide corresponding to the formula R¹⁰OM wherein M isalkali metal and R¹⁰O— corresponds to the alkoxy substituent of R¹, saidcompound of Formula XIV having the structure:

wherein —A—A—, R³, and —B—B—, are as defined above.
 24. A process as setforth in claim 23 wherein said compound of Formula XIV is4′S(4′α),7′α-1′,2′,3′,4,4′,5,5′,6′,7′,8′,10′,12′,13′,14′,15′,16′-hexadecahydro-10β-,13β-dimethyl-3′,5,20′-trioxospiro[furan-2(3H),17′β-[4,7]methano[17H]cyclopenta[a]phenanthrene]5′-carbonitrile.25. A process for the preparation of a compound of Formula XIV:

wherein —A—A— represents the group —CHR⁴—CHR⁵— or —CR⁴═CR⁵— R³, R⁴ andR⁵ are independently selected from the group consisting of hydrogen,halo, hydroxy, lower alkyl, lower alkoxy, hydroxyalkyl, alkoxyalkyl,hydroxycarbonyl, cyano, aryloxy, —B—B— represents the group —CHR⁶—CHR⁷—or an alpha- or beta-oriented group:

 where R⁶ and R⁷ are independently selected from the group consisting ofhydrogen, halo, lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl,hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl, cyano, aryloxy,the process comprising: hydrolyzing a compound corresponding to FormulaXV:

wherein —A—A—, R³, and —B—B— are as defined above.
 26. A process as setforth in claim 25 wherein said compound of Formula XIV is4′S(4′α),7′α-1′,2′,3′,4,4′,5,5′,6′,7′,8′,10′,12′,13′,14′,15′,16′-hexadecahydro-10β-,13′β-dimethyl-3′,5,20′-trioxospiro[furan-2(3H),17′β-[4,7]methano[17H]cyclopenta[a]phenanthrene]5′-carbonitrileand said compound of Formula XV is5′R(5′α),7′β-20′-amino-1′,2′,3′,4,5,6′,7′,8′,10′,12′,13′,14′,15′,16′-tetradecahydro-10′α,13′α-dimethyl-3′,5-dioxospiro[furan-2(3H),17′α(5′H)-[7,4]metheno[4H]cyclopenta[a]phenanthrene]-5′-carbonitrile.
 27. Aprocess for the preparation of a compound corresponding to Formula XV:

wherein —A—A— represents the group —CHR⁴—CHR⁵— or —CR⁴═CR⁵— R³, R⁴ andR⁵ are independently selected from the group consisting of hydrogen,halo, hydroxy, lower alkyl, lower alkoxy, hydroxyalkyl, alkoxyalkyl,hydroxycarbonyl, cyano, aryloxy, —B—B— represents the group —CHR —CHR⁷—or an alpha- or beta-oriented group:

 where R⁶ and R⁷ are independently selected from the group consisting ofhydrogen, halo, lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl,hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl, cyano, aryloxy,the process comprising: reacting a compound of Formula XVI with a sourceof cyanide ion in the presence of an alkali metal salt, said compound ofFormula XVI having the structure:

wherein —A—A—, R³, and —B—B— are as defined above.
 28. A process as setforth in claim 27 wherein said compound of Formula XV is Methyl Hydrogen9α,17α-dihydroxy-3-oxopregn-4-ene-7α,21-dicarboxylate, γ-lactone.
 29. Aprocess for the preparation of a compound corresponding to the formula:

wherein —A—A— represents the group —CHR⁴—CHR⁵— or —CR⁴═CR⁵— R³, R⁴ andR⁵ are independently selected from the group consisting of hydrogen,halo, hydroxy, lower alkyl, lower alkoxy, hydroxyalkyl, alkoxyalkyl,hydroxycarbonyl, cyano, aryloxy, R¹ represents an alpha-oriented loweralkoxycarbonyl or hydroxycarbonyl radical, —B—B— represents the group—CHR⁶—CHR⁷— or an alpha- or beta-oriented group:

 where R⁶ and R⁷ are independently selected from the group consisting ofhydrogen, halo, lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl,hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl, cyano, aryloxy,the process comprising: reacting a compound of Formula XXI with analkali metal alkoxide corresponding to the formula R¹⁰OM wherein M isalkali metal and R¹⁰O— corresponds to the alkoxy substituent of R¹, saidcompound of Formula XXI having the structure:

wherein —A—A— R¹, R³, and —B—B— are as defined above.
 30. A process asset forth in claim 29 wherein said compound of Formula XXI is4′S(4′α),7′α-9′,11α-epoxyhexadecahydro-10β-,13′β-dimethyl-3′5,20′-trioxospiro[furan-2(3H),17′β-4,7]methano[17H]cyclopenta[a]phenanthrene-5′-carbonitrile.31. A process for the preparation of a compound corresponding to FormulaXXI:

wherein —A—A— represents the group —CHR⁴—CHR⁵— or —CR⁴═CR⁵— R³, R⁴ andR⁵ are independently selected from the group consisting of hydrogen,halo, hydroxy, lower alkyl, lower alkoxy, hydroxyalkyl, alkoxyalkyl,hydroxycarbonyl, cyano, aryloxy, —B—B— represents the group —CHR⁶—CHR⁷—or an alpha- or beta-oriented group:

 where R⁶ and R⁷ are independently selected from the group consisting ofhydrogen, halo, lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl,hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl, cyano, aryloxy,the process comprising: hydrolyzing a compound corresponding to FormulaXXII:

wherein —A—A—, R³, and —B—B— are as defined above.
 32. A process as setforth in claim 31 wherein said compound of Formula XXI is4′S(4′α),7′α-9′,11α-epoxyhexadecahydro-10β,13′β-dimethyl-3′5,20′-trioxospiro[furan-2(3H),17′β-[4,7]methano[17H]cyclopenta[a]phenanthrene-5′-carbonitrileand said compound of Formula XXII is5′R(5′α),7′β-20′-amino-9,11β-epoxyhexadecahydro-10′,13′-dimethyl-3′,5-dioxospiro[furan-2(3H),17′α(5′H)-[7,4]methene[4H]cyclopenta[a]phenanthrene-5′-carbonitrile.33. A process for the preparation of a compound corresponding to FormulaXXII:

wherein —A—A— represents the group —CHR⁴—CHR⁵— or —CR⁴═CR⁵— R³, R⁴ andR⁵ are independently selected from the group consisting of hydrogen,halo, hydroxy, lower alkyl, lower alkoxy, hydroxyalkyl, alkoxyalkyl,hydroxycarbonyl, cyano, aryloxy, —B—B— represents the group —CHR⁶—CHR⁷—or an alpha- or beta-oriented group:

 where R⁶ and R⁷ are independently selected from the group consisting ofhydrogen, halo, lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl,hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl, cyano, aryloxy,the process comprising: reacting a compound of Formula XXIII with asource of cyanide ion in the presence of a an alkali metal salt, saidcompound of Formula VIII having the structure:

wherein —A—A—, R³, and —B—B— are as defined above.
 34. A process as setforth in claim 33 wherein said compound of Formula XXII is5′R(5′α),7′β-20′-amino9, 11β-epoxyhexadecahydro-10′,1340-dimethyl-3′,5-dioxospiro[furan-2(3H),17′α(5,′H) -[7,4]methene[4H]cyclopenta[a]phenanthrene-5′-carbonitrile and said compound ofFormula XXII is9,11α-epoxy-17α-hydroxy-3-oxopregna-4,6-diene-21-carboxylic acid,γ-lactone.
 35. A process for the preparation of a compound correspondingto Formula XIII:

wherein —A—A— represents the group —CHR⁴—CHR⁵— or —CR⁴═CR⁵— R³, R⁴ andR⁵ are independently selected from the group consisting of hydrogen,halo, hydroxy, lower alkyl, lower alkoxy, hydroxyalkyl, alkoxyalkyl,hydroxycarbonyl, cyano, aryloxy, R¹ represents an alpha-oriented loweralkoxycarbonyl radical, —B—B— represents the group —CHR⁶—CHR⁷— or analpha- or beta-oriented group:

 where R⁶ and R⁷ are independently selected from the group consisting ofhydrogen, halo, lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl,hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl, cyano, aryloxy,the process comprising: abstracting hydrogen from the 6 and 7 positionsof a compound corresponding to the formula:

wherein —A—A—, R³, and —B—B— are as defined above.
 36. A process for thepreparation of a compound of Formula XIV:

wherein —A—A— represents the group —CHR⁴—CHR⁵— or —CR⁴═CR⁵— R³, R⁴ andR⁵ are independently selected from the group consisting of hydrogen,halo, hydroxy, lower alkyl, lower alkoxy, hydroxyalkyl, alkoxyalkyl,hydroxycarbonyl, cyano, aryloxy, —B—B— represents the group —CHR⁶—CHR⁷—or an alpha- or beta-oriented group:

 where R⁶ and R⁷ are independently selected from the group consisting ofhydrogen, halo, lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl,hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl, cyano, aryloxy,the process comprising: hydrolyzing a compound corresponding to FormulaXXV:

wherein R^(x) is a hydroxyl protecting group and wherein —A—A—, R₁—B—B—,R⁸, and R⁹ are as defined above.
 37. A process as set forth in claim 36wherein said compound of Formula XIV is4′S(4′α),7′α-1′,2′,3′,4,4′,5,5′,6′,7′,8′,10′,12′,13′,14′,15′,16′-hexadecahydro-10β-,13′β-dimethyl-3′,5,20′-trioxospiro[furan-2(3H),17′β-[4,7]methano[17H]cyclopenta[a]phenanthrene]5′-carbonitrileand said compound of Formula XXV is5′R(5′α),7′β-20′-aminohexadecahydro-9′β-hydroxy-10′a,13′α-dimethyl-3′,5-dioxospiro[furan-2(3H),17′α(5′H)-[7,4]metheno[4H]cyclopenta[a]phenanthrene]-5′-carbonitrile.38. A process for the preparation of a compound corresponding to FormulaXXV:

wherein —A—A— represents the group —CHR⁴—CHR⁵— or —CR⁴═CR⁵— R³, R⁴ andR⁵ are independently selected from the group consisting of hydrogen,halo, hydroxy, lower alkyl, lower alkoxy, hydroxyalkyl, alkoxyalkyl,hydroxycarbonyl, cyano, aryloxy, —B—B— represents the group —CHR⁶—CHR⁷—or an alpha- or beta-oriented group:

 where R⁶ and R⁷ are independently selected from the group consisting ofhydrogen, halo, lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl,hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl, cyano, aryloxy,and R⁸ and R⁹ are independently selected from the group consisting ofhydrogen, halo, lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl,hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl, cyano, aryloxy, orR⁸ and R⁹ together comprise a carbocyclic or heterocyclic ringstructure, where R^(x) is a hydroxy protecting group, the processcomprising: reacting a compound of Formula XXVI with a source of cyanideion in the presence of a an alkali metal salt, said compound of FormulaXXVI having the structure:

wherein —A—A—, R³, and —B—B— are as defined above.
 39. A process as setforth in claim 38 wherein said compound of Formula XXV is5′R(5′α),7′β-20′-aminohexadecahydro-9′β-hydroxy-10′a,13′α-dimethyl-3′,5-dioxospiro[furan-2(3H),17′α(5′H)-[7,4]metheno[4H]cyclopenta[a]phenanthrene]-5′-carbonitrileand said compound of Formula XXVI is 9α,17α-dihydroxy-3-oxopregna-4,6-diene-21-carboxylic acid, γ-lactone.
 40. A process forthe preparation of a compound corresponding to Formula XXVI:

wherein —A—A— represents the group —CHR⁴—CHR⁵— or —CR⁴═CR⁵— R³, R⁴ andR⁵ are independently selected from the group consisting of hydrogen,halo, hydroxy, lower alkyl, lower alkoxy, hydroxyalkyl, alkoxyalkyl,hydroxycarbonyl, cyano, aryloxy, —B—B— represents the group —CHR⁶—CHR⁷—or an alpha- or beta-oriented group:

 where R⁶ and R⁷ are independently selected from the group consisting ofhydrogen, halo, lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl,hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl, cyano, aryloxy,where R^(x) is a hydroxy protecting group, the process comprising:abstracting hydrogens from the 6 and 7 positions (dehydrogenation) of acompound corresponding to the formula:

wherein —A—A—, R³, and —B—B— are as defined above.
 41. A process as setforth in claim 40 wherein said compound of Formula XXVI is9α,17α-dihydroxy-3-oxopregna -4,6-diene-21-carboxylic acid, γ-lactoneand said compound of Formula XXVII is 9α,17α-dihydroxy-3-oxopregn-4-ene-21-carboxylic acid, γ-lactone.
 42. A process for the preparationof a compound corresponding to Formula VIII:

wherein —A—A— represents the group —CHR⁴—CHR⁵— or —CR⁴═CR⁵— R³ isselected from the group consisting of hydrogen, halo, hydroxy, loweralkyl, lower alkoxy, hydroxyalkyl, alkoxyalkyl, hydroxycarbonyl, cyano,aryloxy, —B—B— represents the group —CHR⁶—CHR⁷— or an alpha- orbeta-oriented group:

 where R⁶ and R⁷ are independently selected from the group consisting ofhydrogen, halo, lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl,hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl, cyano, aryloxy,and R⁸ and R⁹ are independently selected from the group consisting ofhydrogen, halo, lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl,hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl, cyano, aryloxy orR⁸ and R⁹ together comprise a carbocyclic or heterocyclic ringstructure, or R⁸ and R⁹ together with R⁶ or R⁷ comprise a carbocyclic orheterocyclic ring structure fused to the pentacyclic D ring, the processcomprising: oxidizing a compound of Formula corresponding to Formula 104

wherein —A—A—, R³, and —B—B— are as defined above and R¹¹ is a C₁ to C₄alkyl.
 43. A process as set forth in claim 42 wherein the compound ofFormula VIII is contacted with an oxidizing agent.
 44. A process as setforth in claim 43 wherein said oxidizing agent is a benzoquinonederivative.
 45. A process as set forth in claim 44 wherein saidoxidizing agent is selected from the group consisting of2,3,-dichloro-5,6-dicyano-1,4-benzoquinone and tetrachlorobenzoquinone.46. A process as set forth in claim 42 wherein said compound of Formula104 is contacted with a halogenating agent to produce a halogenatedintermediate; and contacting said halogenated intermediate with adehydrohalogenating agent to dehydrohalogenate said halogenatedintermediate and form said compound of Formula
 104. 47. A process forthe preparation of a compound corresponding to Formula 104:

wherein —A—A— represents the group —CHR⁴—CHR⁵— or —CR⁴═CR⁵— R³ isselected from the group consisting of hydrogen, halo, hydroxy, loweralkyl, lower alkoxy, hydroxyalkyl, alkoxyalkyl, hydroxycarbonyl, cyano,aryloxy, R¹¹ is C₁ to C₄ lower alkyl; —B—B— represents the group—CHR⁶—CHR⁷— or an alpha- or beta-oriented group:

 where R⁶ and R⁷ are independently selected from the group consisting ofhydrogen, halo, lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl,hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl, cyano, aryloxy,the process comprising: thermally decomposing a compound correspondingto Formula 103 in the presence of an alkali metal halide, said compoundof Formula 103 having the structure:

wherein —A—A—, R³, R³, and —B—B— are as defined above and R¹² is C₁-C₄alkyl.
 48. A process for the preparation of a compound corresponding toFormula 103:

wherein —A—A— represents the group —CHR⁴—CHR⁵— or —CR⁴═CR⁵— R³ isselected from the group consisting of hydrogen, halo, hydroxy, loweralkyl, lower alkoxy, hydroxyalkyl, alkoxyalkyl, hydroxycarbonyl, cyano,aryloxy, R¹¹ is C₁-C₄ lower alkyl; —B—B— represents the group—CHR⁶—CHR⁷— or an alpha- or beta-oriented group:

 where R⁶ and R⁷ are independently selected from the group consisting ofhydrogen, halo, lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl,hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl, cyano, aryloxy,the process comprising: condensing a compound of Formula 102 with adialkyl malonate in the presence of a base, said compound of Formula 102having the structure:

wherein —A—A—, R³, R¹¹, and —B—B— are as defined above.
 49. A processfor the preparation of a compound corresponding to Formula 102:

wherein —A—A— represents the group —CHR⁴—CHR⁵— or —CR⁴═CR⁵— R³ isselected from the group consisting of hydrogen, halo, hydroxy, loweralkyl, lower alkoxy, hydroxyalkyl, alkoxyalkyl, hydroxycarbonyl, cyano,aryloxy, R¹¹ is C₁ to C₄ alkyl; —B—B— represents the group —CHR⁶—CHR⁷—or an alpha- or beta-oriented group:

 where R⁶ and R⁷are independently selected from the group consisting ofhydrogen, halo, lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl,hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl, cyano, aryloxy,the process comprising: reacting a compound of Formula 101 with asulfonium ylide in the presence of a base, said compound of Formula 101having the structure:

wherein —A—A—, R³, and —B—B— are as defined above.
 50. A process for thepreparation of a compound corresponding to Formula 101:

wherein —A—A— represents the group —CHR⁴—CHR⁵— or —CR⁴═CR⁵— R³ isselected from the group consisting of hydrogen, halo, hydroxy, loweralkyl, lower alkoxy, hydroxyalkyl, alkoxyalkyl, hydroxycarbonyl, cyano,aryloxy, R¹¹ is C₁-C₄ alkyl; —B—B— represents the group —CHR⁶—CHR⁷— oran alpha- or beta-oriented group:

 where R⁶ and R⁷ are independently selected from the group consisting ofhydrogen, halo, lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl,hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl, cyano, aryloxy,the process comprising: reacting a compound of Formula XXXVI with anetherifying reagent in the presence of an acid catalyst, said compoundof Formula XXXVI having the structure:

wherein —A—A—, R³, and —B—B— are as defined above.
 51. A process as setforth in claim 50 wherein said compound of Formula 101 prepared byreacting a compound of Formula XXXVI with a trialkyl orthoformate in anacidified alkanol solvent.
 52. A process for the preparation of acompound of Formula XXXVI

wherein —A—A— represents the group —CHR⁴—CHR⁵— or —CR⁴═CR⁵— R³ isselected from the group consisting of hydrogen, halo, hydroxy, loweralkyl, lower alkoxy, hydroxyalkyl, alkoxyalkyl, hydroxycarbonyl, cyano,aryloxy, —B—B— represents the group —CHR⁶—CHR⁷— or an alpha- orbeta-oriented group:

 where R⁶ and R⁷ are independently selected from the group consisting ofhydrogen, halo, lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl,hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl, cyano, aryloxy,the process comprising: oxidizing a substrate compound of Formula XXXVIIby fermentation in the presence of a microorganism effective forconversion of said substrate compound to a compound of Formula XXXVI

where —A—A—, —B—B— and R³ are as defined above, said substrate compoundof Formula XXXVII corresponding to the Formula:

wherein —A—A—, R¹, R³, —B—B—, and are as defined above and D—D is—CH₂—CH₂— or —CH═CH— and R¹³, R¹⁴, R¹⁵, and R¹⁶ are independentlyselected from the group consisting of C₁-C₄ alkyl; and thereafterintroducing an 11-hydroxy group into said α-orientation in said compoundof Formula XXXVI by fermentation in the presence of a microorganismeffective for the 11α-hydroxylation.
 53. A process for the preparationof a compound corresponding to Formula II:

wherein: —A—A— represents the group —CHR⁴—CHR⁵— or —CR⁴═CR⁵— R³, R⁴ andR⁵ are independently selected from the group consisting of hydrogen,halo, hydroxy, lower alkyl, lower alkoxy, hydroxyalkyl, alkoxyalkyl,hydroxycarbonyl, cyano, aryloxy, R¹ represents an alpha-oriented loweralkoxycarbonyl or hydroxycarbonyl radical, —B—B— represents the group—CHR⁶—CHR⁷— or an alpha- or beta-oriented group:

 where R⁶ and R⁷ are independently selected from the group consisting ofhydrogen, halo, lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl,hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl, cyano, aryloxy,and R⁸ and R⁹ are independently selected from the group consisting ofhydrogen, halo, lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl,hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl, cyano, aryloxy, orR⁸ and R⁹ together comprise a carbocyclic or heterocyclic ringstructure, the process comprising: preparing a compound of Formula V

 wherein —A—A—, R¹, R³, —B—B—, R⁸, and R⁹ are as defined above byreacting a compound of Formula VI with an alkali metal alkoxidecorresponding to the formula R¹⁰OM wherein M is alkali metal and R¹⁰O—corresponds to the alkoxy substituent of R¹, said compound of Formula VIhaving the structure:

 wherein —A—A—, R³, —B—B—, R⁸, and R⁹ are as defined above; withoutisolating said compound of Formula V in purified form, reacting saidcompound of Formula V with a lower alkylsulfonylating or acylatingreagent to produce a compound of Formula IV

 wherein —A—A—, R¹, R³, —B—B—, R⁸, and R⁹ are as defined above, and R²is alkylsulfonyloxy, acyloxy leaving group or halide; without isolatingsaid compound of Formula IV in purified form, removing the 11α-leavinggroup therefrom by reaction with a reagent for abstraction thereof toproduce said compound of Formula II.
 54. A process as set forth in claim53 wherein, without isolating said compound of Formula II in purifiedform, said compound of Formula II is reacted with an epoxidizing reagentto form a product of Formula I

wherein —A—A—, R¹, R³, —B—B—, R⁸, and R⁹ are as defined above.
 55. Aprocess as set forth in claim 54 wherein: said compound of Formula II isformed by reaction of said compound of Formula IV with a leaving groupremoving reagent comprising an alkanoic acid in the presence of analkali metal alkoxide; volatile components are stripped from thereaction solution; water-soluble components of the reaction solution areremoved by washing with an aqueous washing solution, thereby producingresidual Formula II solution suitable for conversion of the compound ofFormula II to a compound of Formula I; and a peroxide oxidizing agent iscombined with the residual Formula II solution to effect the conversionof the compound of Formula II to the compound of Formula I.
 56. Aprocess as set forth in claim 54 wherein: said compound of Formula V isformed by reaction of said compound of Formula VI with an alkali metalalkoxide in an organic solvent; the compound of Formula V is extractedfrom a solution comprising the Formula V reaction solution using anorganic solvent, thereby producing a Formula V extract solution; and alower alkylsulfonyl halide or acyl halide is introduced into a solutioncomprising said Formula V extract solution for preparation of thecompound of Formula VI.
 57. A process as set forth in claim 54 wherein:said compound of Formula IV is formed by reaction of said compound ofFormula V with a leaving group abstraction reagent in an organicsolvent; a solution comprising the Formula IV reaction solution ispassed over an acidic and then a basic exchange resin column for theremoval of basic and acidic impurities therefrom, thereby producingFormula IV eluate solution; and a reagent for abstraction of analkylsulfonyloxy or acyloxy leaving group is combined with a solutioncomprising said Formula IV eluate solution for preparation of saidcompound of Formula II.
 58. A process for the formation of an epoxycompound comprising contacting a substrate compound having an olefinicdouble bond with a peroxide compound in the presence of a peroxideactivator, said peroxide activator corresponding to the formula:

where R is a substituent having an electron withdrawing strength notless than that monochloromethyl.
 59. A process as set forth in claim 58wherein said peroxide activator corresponds to the formula

where X¹, X², and X³ are selected from the group consisting of halo,hydrogen, alkyl, haloalkyl, cyano and cyanoalkyl, R^(P) is selected fromthe group consisting of arylene and —(CX⁴X⁵)_(n)—, and n is 0, or 1, atleast one of X¹, X², X³, X⁴ and X⁵ being halo or perhaloalkyl.
 60. Aprocess as set forth in claim 58 wherein n is 0 and at least two of X¹,x² and X³ are halo or perhaloalkyl.
 61. A process as set forth in claim58 wherein all of X¹, X², X³, X⁴ and X⁵ are halo or perhaloalkyl.
 62. Aprocess as set forth in claim 58 wherein said peroxide activator is atrihaloacetamide.
 63. A process as set forth in claim 62 wherein saidperoxide activator is trichloroacetamide.
 64. A process as set forth inclaim 58 wherein said substrate compound corresponds to the Formula:

wherein —A—A— represents the group —CHR⁴—CHR⁵— or —CR⁴═CR⁵— R³, isselected from the group consisting of hydrogen, halo, hydroxy, loweralkyl, lower alkoxy, hydroxyalkyl, alkoxyalkyl, hydroxy carbonyl, cyano,aryloxy, R¹ represents an alpha-oriented lower alkoxycarbonyl orhydroxycarbonyl radical, —B—B— represents the group —CHR⁶—CHR⁷— or analpha- or beta-oriented group:

 where R⁶ and R⁷ are independently selected from the group consisting ofhydrogen, halo, lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl,hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl, cyano, aryloxy,and R⁸ and R⁹ are independently selected from the group consisting ofhydrogen, halo, lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl,hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl, cyano, aryloxy, orR⁸ and R⁹ together comprise a carbocyclic or heterocyclic ringstructure, or R⁸ or R⁹ together with R⁶ or R⁷ comprise a carbocyclic orheterocyclic ring structure fused to the pentacyclic D ring.
 65. Aprocess as set forth in claim 58 wherein said substrate compound isselected from the group consisting of:

and the product of the epoxidation reaction is selected from the groupconsisting of: